Clinical proteomics: study of a cryogel

Cryoproteins are proteins precipitating at low temperature. Usually, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Very rarely, Igs do not precipitate, but, upon cooling, form a gel. Here, we report a case of cryogel observed in a patient presenting with Waldenström's disease. Using proteomic tools, a monoclonal IgM was identified as being the cause of the gel formation. Furthermore, addition of H₂O before incubation at 4°C demonstrated that the monoclonal IgM was precipitable as a type I cryoglobulin (hypocryoglobulin).     

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.     

Chromatin boundaries in budding yeast: the nuclear pore connection

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.     

Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow.

The adhesion of moving cells to receptor-bearing surfaces is a key step to many important biological processes. Attachment was subjected to extensive modeling. However, the numerical values of kinetic bonding parameters relevant to realistic models of cell adhesion remain poorly known. In this report, we describe the motion of human granulocytes to interleukin-1-activated endothelial cells in presence of a low hydrodynamic drag (a few piconewtons) estimated to be much weaker than a standard ligand-receptor bond. It was thus expected to visualize the formation and rupture of individual bonds. We observed multiple short-time cell arrests with a median duration of 2.43 s. Stop frequency, not duration, was significantly inhibited by anti-E-selectin antibodies. Binding efficiency exhibited an almost linear relationship with the inverse of cell velocity. The distribution of arrest duration was determined: results were consistent with the view that these arrests reflected the formation/dissociation of single ligand-receptor bonds with a spontaneous dissociation rate of 0.5 s-1. The rate of bond formation was on the order of 0.04 s-1 when cells were freely rolling (mean velocity: 19 microns/s) and it exhibited an approximately 10-fold increase after the formation of a first adhesion.