Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.     

The relation of the microglia with the pericytes in the cat cerebral cortex

Summary Capillaries, pericytes and microglial cells in layer I of the cerebral cortex of normal adult cats have been studied with electron microscopy. The data obtained in this study show that pericytes are cells which are able to transform themselves into microglial cells by virtue of an activation process in which the astrocytic neuroglia appears to play a decisive role. By virtue of its structure, its mesodermic origin and its function the microglia has to be distinguished clearly from the astrocytic neuroglia and the oligodendroglia.This study was partly supported by a grant from the Seguridad Social, Instituto Nacional de Previsión.     

Enhanced cytoplasmic sequestration of the nuclear export receptor CRM1 by NS2 mutations developed in the host regulates parvovirus fitness

To investigate whether a DNA virus can evade passive immunotherapy with a polyclonal antiserum, we analyzed the protection of a neutralizing capsid antiserum against a lethal infection of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) in 42 immunodeficient mice over a period of 200 days. A few mice were effectively protected, but most developed a delayed lethal leukopenic syndrome during the treatment or weeks afterwards. Unexpectedly, viruses isolated from treated but also from control leukopenic mice showed no amino acid changes throughout the entire capsid coding region, although the viral populations were genetically heterogeneous, mainly in the second exon of the coding sequence of the NS2 nonstructural protein. The NS2 point amino acid changes (T88A, K96E, L103P, and L153 M) that were consistently selected in several mice clustered within the nuclear exportin CRM1 binding domain, in a reading frame that did not alter the overlapping NS1 coding region. These mutations endowed emerging viruses with an increased fitness that was demonstrable by their relative resistance to the neutralizing capsid antiserum in a postentry plaque-forming assay, the rapid overgrowth of a competing wild-type (wt) population in culture, and a larger yield of infectious particles. Mutant NS2 proteins interacted with a higher affinity and sequestered CRM1 in the perinuclear region of the cytoplasm more efficiently than the wt. Correspondingly this phenomenon, as well as the following timely ordered release of the NS1 nonstructural protein and the empty capsid from the nucleus to the cytoplasm, occurred markedly earlier in the infection cycle of the mutant viruses. We hypothesize that the enhanced cytoplasmic sequestration of CRM1 by the NS2 mutations selected in mice may trigger pleiotropic effects leading to an accelerated MVMi life cycle and thus to increased fitness. These results strengthen our earlier report on the rapid evolutionary capacity of this mammalian-specific DNA virus in vivo and indicate that the NS2-CRM1 interaction is an important determinant of parvovirus virulence that can be modulated in nature, hampering the effectiveness of passive antibody therapies in the long term.     

A Novel,Single Algorithm Approach to Predict Acenocoumarol Dose Based on CYP2C9 and VKORC1 Allele Variants

The identification of CYP2C9 and VKORC1 genes has strongly stimulated the research on pharmacogenetics of coumarins in the last decade. We assessed the combined influence of CYP2C9 *2 and *3, and VKORC1 c.-1639G>A, 497C>G, and 1173C>T variants, on acenocoumarol dosage using a novel algorithm approach, in 193 outpatients who had achieved stable anticoagulation. We constructed an “acenocoumarol-dose genotype score” (AGS, maximum score = 100) based on the number of alleles associated with higher acenocoumarol dosage carried by each subject for each polymorphism. The mean AGS was higher in the high-dose (>28mg/week) compared with the low-dose (<7mg/week) group (mean(SEM) of 84.1±3.4 vs. 62.2±4.8, P = 0.008). An AGS>70 was associated with an increased odds ratio (OR) of requiring high acenocoumarol dosage (OR: 3.347; 95%CI: 1.112–10.075; P = 0.032). In summary, although more research is necessary in other patient cohorts, and this algorithm should be replicated in an independent sample, our data suggest that the AGS algorithm could be used to help discriminating patients requiring high acenocoumarol doses to achieve stable anti-coagulation.     

BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions

Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b(-/-) mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b(-/-) mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT.     

Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity

The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions. Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.     

Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.     

In vitro Culture of Medicago arborea L. Anthers: Initial Response

High production of viable somatic embryos was obtained from cultured anthers in the second phase of meiosis, using microscopic level observations of tetrads. The medium with the greatest embryogenic efficiency was H6, composed of Murashige and Skoog (MS) medium with 2 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l⁻¹ of kinetin. All (100%) of the somatic embryos obtained germinated and produced 63% green and 37% albino seedlings. In general, embryogenic calli had a higher ion concentration than non-embryogenic calli, with the exception of calcium whose concentration was higher in non-embryogenic calli. The calli induced in the different media differed in their sucrose and starch compositions. The most embryogenic medium H6-induced calli with the highest sucrose concentration and the lowest starch concentration, before visible embryos were observed. In the leaves of the albino seedlings, sucrose concentrations were very high while those of starch were very low. Ion concentrations were also lower in albino plants than in the leaves of green seedlings, with the exception of calcium, whose concentration was higher. Most of the albino individuals were homozygous, even when their progenitors were heterozygous, thereby confirming their haploid nature.