A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.     

Genotype-specific interactions and the trade-off between host and parasite fitness

Background  

Evolution of parasite traits is inextricably linked to their hosts. For instance one common definition of parasite virulence is the reduction in host fitness due to infection. Thus, traits of infection must be viewed in both protagonists and may be under shared genetic and physiological control. We investigated these questions on the oomycete Hyaloperonospora arabidopsis (= parasitica), a natural pathogen of the Brassicaceae Arabidopsis thaliana.     

Anthocyanins of the Sterculiaceae: Flavonoid scores and Hennigian phylogenetic systematics

Published results of the distribution of anthocyanins in the Sterculiaceae have been re-interpreted on the basis of the phylogenetic status of the compounds present. A flavonoid score system was less useful than a cladistic interpretation based on Hennigian arguments.     

Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis

Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M. tuberculosis, the cell wall mycoloylation decreases by 40% when fbpC is knocked out. To investigate whether the remaining 60% mycoloylation came from the activity of FbpA and/or FbpB, fbpA- and fbpB-inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC-disrupted mutant. Unexpectedly, both mutants produced normally mycoloylated cell walls. Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC-inactivated mutant strain of M. tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC-inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions. Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non-reducing end hexaarabinosides.     

Glomulin is predominantly expressed in vascular smooth muscle cells in the embryonic and adult mouse

Mutations in the glomulin gene result in dominantly inherited vascular lesions of the skin known as glomuvenous malformations (GVMs). These lesions are histologically distinguished by their distended vein-like channels containing characteristic 'glomus cells', which appear to be incompletely or improperly differentiated vascular smooth muscle cells (VSMCs). The function of glomulin is currently unknown. We studied glomulin expression during murine development (E9.5 days post-coitum until adulthood) by non-radioactive in situ hybridization. Glomulin was first detected at E10.5 dpc in cardiac outflow tracts. Later, it showed strong expression in VSMCs as well as a limited expression in the perichondrium. At E11.5-14.5 dpc glomulin RNA was most abundant in the walls of the large vessels. At E16.5 dpc expression was also detectable in smaller arteries and veins. The high expression of glomulin in murine vasculature suggests an important role for glomulin in blood vessel development and/or maintenance, which is supported by the vascular phenotype seen in GVM patients with mutations in this gene.     

Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.     

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

Background  

Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization.     

Homoeologous recombination within bread wheat to develop novel combinations of HMW-GS genes: transfer of the <Emphasis Type="Italic">Glu-A1</Emphasis> locus to chromosome 1D

In an attempt to improve the bread-making quality within hexaploid wheat by elaborating novel high-molecular weight glutenin subunits (HMW-GS) combinations useful in wheat-breeding programmes, a 1A chromosome fragment carrying the Glu-A1 locus encoding the subunit Ax2*, was translocated to the long arm of chromosome 1D. The partially isohomoeoallelic line, designated RR239, had a meiotic behaviour as regular as cv. Courtot. It was characterised using genomic in situ hybridization and microsatellite markers as well as biochemical and proteomic approaches. The translocated 1D chromosome had an interstitial 1AL segment representing in average 30% of the recombinant arm length that was confirmed by molecular analysis. The genetic length of the removed segment in chromosome 1DL was estimated to be at least 51 cM, and that of the interstitial 1AL translocation to be at least 33 cM. Proteome analysis performed on total endosperm proteins revealed variation in amounts, 8 spots and 1 spot being up- and downregulated, respectively. Quantitative variations in HMW-GS were observed for the Glu-A1 (Ax2*) and Glu-B1 (Bx7 + By8) loci in response to duplication of the Glu-A1 locus.     

Multimeric BLM is dissociated upon ATP hydrolysis and functions as monomers in resolving DNA structures

Bloom (BLM) syndrome is an autosomal recessive disorder characterized by an increased risk for many types of cancers. Previous studies have shown that BLM protein forms a hexameric ring structure, but its oligomeric form in DNA unwinding is still not well clarified. In this work, we have used dynamic light scattering and various stopped-flow assays to study the active form and kinetic mechanism of BLM in DNA unwinding. It was found that BLM multimers were dissociated upon ATP hydrolysis. Steady-state and single-turnover kinetic studies revealed that BLM helicase always unwound duplex DNA in the monomeric form under conditions of varying enzyme and ATP concentrations as well as 3′-ssDNA tail lengths, with no sign of oligomerization being discerned. Measurements of ATPase activity further indicated that BLM helicase might still function as monomers in resolving highly structured DNAs such as Holliday junctions and D-loops. These results shed new light on the underlying mechanism of BLM-mediated DNA unwinding and on the molecular and functional basis for the phenotype of heterozygous carriers of BLM syndrome.