The Glycine-Rich Motif of Pyrococcus abyssi DNA Polymerase D Is Critical for Protein Stability

A glycine-rich motif described as being involved in human polymerase δ proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D.     

Corticosteroid effects on ventilator-induced diaphragm dysfunction in anesthetized rats depend on the dose administered

Background

High dose of corticosteroids has been previously shown to protect against controlled mechanical ventilation (CMV)-induced diaphragmatic dysfunction while inhibiting calpain activation. Because literature suggests that the calpain inhibiting effect of corticosteroid depends on the dose administered, we determined whether lower doses of corticosteroids would also provide protection of the diaphragm during CMV. This may be important for patients undergoing mechanical ventilation and receiving corticosteroids.

Methods

Rats were assigned to controls or to 24 hours of CMV while being treated at the start of mechanical ventilation with a single intramuscular administration of either saline, or 5 mg/kg (low MP) or 30 mg/kg (high MP) of methylprednisolone.

Results

Diaphragmatic force was decreased after CMV and this was exacerbated in the low MP group while high MP rescued this diaphragmatic dysfunction. Atrophy was more severe in the low MP group than after CMV while no atrophy was observed in the high MP group. A significant and similar increase in calpain activity was observed in both the low MP and CMV groups whereas the high dose prevented calpain activation. Expression of calpastatin, the endogenous inhibitor of calpain, was decreased in the CMV and low MP groups but its level was preserved to controls in the high MP group. Caspase-3 activity increased in all CMV groups but to a lesser extent in the low and high MP groups. The 20S proteasome activity was increased in CMV only.

Conclusions

Administration of 30 mg/kg methylprednisolone during CMV protected against CMV-induced diaphragm dysfunction while 5 mg/kg was more deleterious. The protective effect is due mainly to an inhibition of the calpain system through preservation of calpastatin levels and to a lesser extent to a caspase-3 inhibition.     

Parasite ligand-host receptor interactions during invasion of erythrocytes by Plasmodium merozoites

Malaria parasites must recognise and invade different cells during their life cycle. The efficiency with which Plasmodium falciparum invades erythrocytes of all ages is an important virulence factor, since the ability of the parasite to reach high levels of parasitemia is often associated with severe pathology and morbidity. The merozoite invasion of erythrocytes is a highly complex, multi-step process that is dependent on a cascade of specific molecular interactions. Although many proteins are known to play an important role in invasion, their functional characteristics remain unclear. Therefore, a complete understanding of the molecular interactions that are the basis of the invasion process is absolutely crucial, not only in improving our knowledge about the basic biology of the malarial parasite, but also for the development of intervention strategies to counter the disease. Here we review the current state of knowledge about the receptor-ligand interactions that mediate merozoite invasion of erythrocytes.     

Altered myogenesis in Six1-deficient mice

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1(-/-) embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1(-/-) embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.     

The Arabidopsis MEI1 gene encodes a protein with five BRCT domains that is involved in meiosis-specific DNA repair events independent of SPO11-induced DSBs

Arabidopsis thaliana MEI1 was first described as a gene involved in male meiosis, encoding a short protein showing homology with a human acrosin-trypsin inhibitor. We have isolated a new allele of mei1, and shown that in both mutants male and female meiosis are affected. In both reproductive pathways, meiosis proceeds while chromosomes become fragmented, resulting in aberrant meiotic products and in a strongly reduced fertility. We have shown that the gene mutated in mei1 mutants actually encodes a protein of 972 amino acids that contains five BRCA1 C-terminus (BRCT) domains and is similar to proteins involved in the response to DNA damage and replication blocks in eukaryotes. During meiosis, recombination is initiated by the formation of DNA double strand breaks (DSBs) induced by the protein SPO11. We analysed meiotic chromosome behaviour of the mei1 mutant in a spo11 mutant background and proved that the meiotic fragmentation observed in mei1 mutants was not the consequence of defects in the repair of meiotic DSBs induced by SPO11. We also analysed the effect of mei1 on the mitotic cell cycle but could not detect any sensitivity of mei1 seedlings to DNA-damaging agents like gamma-rays or UV. Therefore, MEI1 is a BRCT-domain-containing protein that could be specific to the meiotic cell cycle and that plays a crucial role in some DNA repair events independent of SPO11 DSB recombination repair.     

Simple fluorescent PCR assay for discriminating FRAXA fully mutated females from normal homozygotes

Fragile X syndrome linked to the FRAXA locus is the most common inherited genetic disease accounting for mental retardation and is usually caused by the expansion of an unstable CGG repeat in the first exon of the FMR1 gene on the X chromosome. Despite its robustness, Southern blot is not suitable for large-scale routine screening as part of neuropediatric practice. PCR appears as an interesting alternative, and various protocols have been successfully applied to molecular screening in mentally retarded boys and girls. Unfortunately, as of this date these protocols are unable to detect the expanded allele in FRAXA females reliably, thereby failing to discriminate between fully mutated females from normal homozygotes. Therefore, we opted for an alternative approach in designing a semiquantitative PCR assay, based on the amplification of the sole wild-type allele. This method allowed us to detect the presence of one or two normal alleles with the same sizes, thereby discriminating between a FRAXA fully mutated female or a normal homozygote, respectively. A trial on 95 DNA samples from normal and mutated females demonstrated the reliability of the procedure. We believe this simple PCR assay is a powerful approach that would reduce the recourse to Southern blotting in females with mental retardation of unknown etiology.