Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.     

Nucleotide mapping and a kinetic model of a heteroplasmic deletion of 4,666 base pairs from mitochondrial DNA in the Kearns-Sayre syndrome

We report the nucleotidic mapping of a 4,666 base pairs deletion of the human mitochondrial DNA localized at positions 8571 and 13237 in a Kearns-Sayre syndrome patient. The gene fusion between the 15 N terminal amino acid residues of ATP synthetase subunit 6 and the 303 C terminal aminoacids of NADH dehydrogenase yields a potential protein of 35,000 d MW called A6-ND5. Deletion boundaries show a short inverted repeat ATCXTA. The heteroplasmic deletion mechanism is discussed in view of these data.     

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome‐like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin‐1 interacted directly with the RuvbL1 barrel‐like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.     

Slow oscillations as a probe of the dynamics of the locus coeruleus-frontal cortex interaction in anesthetized rats

Multiunit or single unit activity recorded simultaneously from frontal cortex (FC) and locus coeruleus (LC) under ketamine anesthesia revealed that both regions show slow oscillatory activity, together or separately. If, however, both regions are engaged in this oscillatory activity, there is a systematic relationship between their phases with peak LC firing always following FC firing by 200–400 ms. This was confirmed by cross-correlational analyses, which indicated that the two structures temporarily form a resonant system. The FC-LC resonant state is, however, loose enough to remain open to other intrinsic or extrinsic influences, keeping the measured frequencies of oscillations at each site slightly different, as demonstrated by a delailed analysis of the autocorrelograms. An injection of lidocaine at the frontal cortex site, while sharply reducing the prefrontal activity to essentially zero, leads to an increase of the LC activity and to a modification of the shape of the LC autocorrelogram, but does not change appreciably the phase relationship between the activity in the two structures during the diminishing activity in FC.     

Inhibition of human leukocyte elastase by acetyl and trifluoroacetyl oligopeptide chloromethyl ketones.

The action of human leukocyte elastase on a series of acetyl and trifluoroacetyl tri-, tetra-, and pentapeptide chloromethyl ketones has been investigated. Leukocyte and pancreatic elastases react quite differently with these irreversible inhibitors. For instance, leukocyte elastase has a much lower affinity for the compounds than pancreatic elastase. On the other hand, the inhibition rate constants of the two enzymes are not influenced in the same way by peptide chain elongation. The two elastases, however, share a common property: trifluoroacetyl tri- and tetraalanine chloromethyl ketones are more tightly bound but are less reactive than the corresponding acetylated inhibitors. This behavior is probably due to the formation of nonproductive complexes between the enzymes and the trifluoroacetylated inhibitors.     

The specificity of aminoacylation: a tRNA-tRNA interaction model.

Most of the isoacceptor species for a particular tRNA can be classified according to the middle base in the anticodon together with the fourth base in the amino acid stem. These specifying nucleotides would operate if a tRNA-tRNA interaction occurs on the aminoacyl-tRNA synthetase so that the anticodon of one tRNA molecule faces the fourth base of the other tRNA molecule. This model explains most of the misacylation reactions or changes in aminoacylation after mutation or chemical modifications of tRNAs. It also provides an explanation for biochemical properties of the aminoacyl-tRNA synthetases such as the presence of two active sites, and for the high fidelity of the aminoacylation. It may give insight into the origin and stability of the genetic code.     

The inhibition of human leukocyte elastase by basic pancreatic trypsin inhibitor

The effects of 30-min intravenous infusions of ethanol (about 50 mm blood concentration), acetaldehyde (about 100 μm blood concentration), and acetate (equimolar dose to acetaldehyde) were studied in normal and adrenalectomized rats. Blood glucose, plasma free fatty acids (FFA), plasma immunoreactive insulin, and glucagon and hepatic glycogen concentrations were measured. Ethanol itself in the presence of 4-methylpyrazole (4-MP) produced no marked changes in the parameters measured. Its infusion without 4-MP reduced plasma insulin by 35% in the normal rats, but not in the adrenalectomized rats, with no simultaneous changes in blood glucose. Acetaldehyde infusion produced hyperglycemia and relatively slight hyperinsulinemia in the normal rats, but not in the adrenalectomized rats. Equimolar acetate was not as potent a stimulator of glycogenolysis as acetaldehyde. Plasma FFA concentrations were markedly reduced by ethanol (without 4-MP), acetaldehyde and acetate both in the normal and adrenalectomized rats, but in the presence of 4-MP ethanol was without effect. The results indicate that metabolites of ethanol (mostly acetaldehyde) produced during ethanol oxidation in vivo are responsible for the stimulation of glycogenolysis through the release of catecholamines from the adrenal glands. The ethanol-induced decrease in plasma FFA is also attributable to the metabolites of ethanol, acetaldehyde having a more potent depressing action than acetate. The mode of inhibition of lipolysis is not related to hormonal factors.     

Evidence for a direct role of the DNA polymerase gamma in the replication of the human mitochondrial DNA in vitro

Indirect experiments suggest that DNA polymerase gamma is involved in the mitochondrial DNA replication process. This report describes an in vitro mitochondrial DNA replication assay directed by the origin of replication of the Heavy strand mt DNA. The assay requires all four dNTP, rNTP and an ATP regenerating system. Nuclease digestion experiments show that specific events occur at the mt origin of replication. Antibodies raised against the purified DNA polymerase gamma inhibit the DNA replication reaction.