Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy.

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.

A reiterative mode of DNA synthesis adopted by HIV-1 reverse transcriptase after a misincorporation.

Amplification of oligonucleotide repeats is a major cause of variability and instability of genomes. This phenomenon is probably due to an aberration in the copying process of polymerases. We show here that in the presence of MnCl2, mismatch formation commits HIV-1 reverse transcriptase to a new mode of DNA synthesis which generates repetitive products. This activity is distinct from terminal transferase since it requires specific DNA motifs in the template. This mechanism, which is processive, also works on homologous RNA templates where it generates reiterative products more than 150 nucleotides long. The corresponding mechanism, which involves extensive primer misalignment, is strikingly similar to that postulated for telomerases.

Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates.

Retroviruses are known to mutate at high rates. An important source of genetic variability is recombination taking place during reverse transcription of internal regions of the two genomic RNAs. We have designed an in vitro model system, involving genetic markers carried on two RNA templates, to allow a search for individual recombination events and to score their frequency of occurrence. We show that Moloney murine leukemia virus reverse transcriptase alone promotes homologous recombination efficiently. While RNA concentration has little effect on recombination frequency, there is a clear correlation between the amount of reverse transcriptase used in the assay and the extent of recombination observed. Under conditions mimicking the in vivo situation, a rate compatible with ex vivo estimates has been obtained.

Comparison of interactions of 5'-derivatives of deoxyoctathymidylate with human DNA polymerize alpha and HIV reverse transcriptase.

Km and Vmax values for d(pT8) and its derivatives containing various 5'-end groups were estimated in the reaction of DNA polymerization alpha catalyzed by DNA polymerase alpha and HIV-RT. The effect of 5'-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT8 is 200-fold higher than that of d(pT8). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase alpha the influence of modifiers on primer affinity is much weaker. The effect of 5'-end residues on the Vmax values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.

E. coli DNA polymerase I as a reverse transcriptase.

The ability of Escherichia coli DNA polymerase I to retrotranscribe an RNA template was examined under steady-state conditions, using a primer extension assay which allows determination of kinetic constants on well-defined heterogeneous sequences. Equilibrium and rate constants for the initial binding step of the enzyme to two homologous DNA and RNA templates do not show striking differences. In both cases, under steady-state conditions, processivity limits the maximal velocity of the translocation process. The lower catalytic efficiency of the enzyme when it operates on RNA is then reflected by a 100-fold greater apparent average Michaelis constant for the deoxynucleotide substrates. We conclude that E.coli DNA polymerase I effectively transcribes both templates, its performances being limited in both cases by its intrinsically low processivity. Furthermore, DNA polymerase I is a strikingly accurate enzyme when operating on RNA. Magnesium has to be substituted by manganese so that a pattern of errors could be detected. This great accuracy results from a combination of factors. The 3' to 5' exonuclease activity is still operating but in a non-discriminative manner. Elongation of a mismatched primer terminus is markedly impaired. The forward polymerization rate of incorporation of an incorrect deoxynucleotide must be extremely low, when Mg2+ is present. In summary E.coli DNA polymerase I preserves its main characteristics when retrotranscribing RNA.

Reverse transcriptases and genomic variability: the accuracy of DNA replication is enzyme specific and sequence dependent.

Kinetics of incorporation of correct and incorrect deoxynucleotides by three reverse transcriptases have been followed, by gel assay, on a series of DNA templates, including part of the HIV-1 gag DNA minus strand. Insertion kinetics for the properly matched nucleotide at a given place on the template vary strongly from one enzyme to the next. No significant correlation is found between the site-specific Michaelis constants, while the maximal velocities are more closely connected. For a given reverse transcriptase these parameters are strongly influenced by the DNA sequence. A systematic evaluation of the frequencies of misincorporation was then performed at 46 positions. Again great variability was found, precluding a very accurate evaluation of an average misincorporation frequency for a given enzyme and a given mismatch. Qualitatively however, HIV-1 reverse transcriptase is certainly not more error-prone in this assay than the other enzymes assayed. The patterns of misincorporations were again very dependent on the enzyme used to replicate a given template. The variability of the gag sequence observed in vivo among various HIV-1 isolates was compared with the patterns of misincorporations obtained in vitro on the same sequence with HIV-1, AMV and MoMLV reverse transcriptases. A fair agreement was found with the pattern observed in the polymerization directed by the HIV-1 reverse transcriptase. The correlation is less important in the two other cases. However some specific changes observed in vivo cannot be accounted for by our misincorporation assay, even when performed with the homologous enzyme, suggesting that an important class of mismatches can only be generated during reverse transcription of the RNA strand. Additional data, using a complementary DNA (positive) strand as a gag template support this hypothesis.

DNA-dependent RNA polymerase of Escherichia coli induces bending or an increased flexibility of DNA by specific complex formation.

Escherichia coli RNA polymerase is shown to induce bending or an increased flexibility of the promoter DNA. This is a specific effect of holoenzyme (core enzyme and sigma-factor). The centre of the flexibility is 3 bp upstream of the initiation point of RNA synthesis. This flexibility or bending is maintained during RNA synthesis by core enzyme.

One-dimensional diffusion of Escherichia coli DNA-dependent RNA polymerase: a mechanism to facilitate promoter location.

The mechanism of promoter location by DNA-dependent RNA polymerase of Escherichia coli was investigated. The occupancies of DNA fragments carrying the A1 promoter of bacteriophage T7 were analyzed as a function of the length of flanking sequences adjacent to the promoter. Competition between the promoters on different fragments showed qualitatively that DNA sequences downstream of the promoter enhanced promoter occupancy, whereas upstream flanking sequences had little or no influence on occupancy. This was studied quantitatively by using a set of DNA fragments with four identical A1 promoters (I-IV) equidistant from each other, but with different lengths of flanking sequences upstream from promoter I and downstream from promoter IV. The relative occupancies of these promoters showed that downstream DNA sequences of up to 250 base pairs increased the occupancy of the adjacent promoter, whereas upstream sequences longer than 70 base pairs had little or no effect on occupancy. Promoter occupancies measured as a function of the length of the downstream flanking DNA sequences were fit by a published theory that takes into account an enhancement of signal-sequence location by linear diffusion.

Piliated Bacteroides fragilis strains adhere to epithelial cells and are more sensitive to phagocytosis by human neutrophils than nonpiliated strains.

Fifteen unencapsulated B. fragilis strains isolated from human infections were examined for their capability to hemagglutinate erythrocytes of different species. Seven strains were found to hemagglutinate guinea pig and human (A,B,O) erythrocytes. This hemagglutination was resistant to treatment with D-mannose and several other sugars. Hemagglutinating strains were also capable of adhering to human epithelial cells and cultured human cell line (Intestine 407) and were 6- to 20-fold more adhesive than non-hemagglutinating strains. Pilus-like structures were found in negative-stained preparations on the hemagglutinating (and adhesive) strains but not on the others. Hemagglutinating and adhesive bacteria were 3- to 7-fold more sensitive to phagocytosis and 5- to 10-fold more sensitive to killing by human neutrophils than non-hemagglutinating ones.