Confirmation and refinement of the genetic localization of the Coffin-Lowry syndrome locus in Xp22.1-p22.2

The Coffin-Lowry syndrome (CLS) is an X-linked inherited disease of unknown pathogenesis characterized by severe mental retardation, typical facial and digital anomalies, and progressive skeletal deformations. Our previous linkage analysis, based on four pedigrees with the disease, suggested a localization for the CLS locus in Xp22.1-p22.2, with the most likely position between the marker loci DXS41 and DXS43. We have now extended the study to 16 families by using seven RFLP marker loci spanning the Xp22.1-p22.2 region. Linkage has been established with five markers from this part of the X chromosome: DXS274 (lod score [Z] (theta) = 3.53 at theta = .08), DXS43 (Z(theta) = 3.16 at theta = .08), DXS197 (Z(theta) = 3.03 at theta = .05), DXS41 (Z(theta) = 2.89 at theta = .08), and DXS207 (Z(theta) = 2.73 at theta = .13). A multipoint linkage analysis further placed, with a maximum multipoint Z of 7.30, the mutation-causing CLS within a 7-cM interval defined by the cluster of tightly linked markers (DXS207-DXS43-DXS197) on the distal side and by DXS274 on the proximal side. Thus, these further linkage data confirm and refine the map location for the gene responsible for CLS in Xp22.1-p22.2. As no linkage heterogeneity was detected, this validates the use of the Xp22.1-p22.2 markers for carrier detection and prenatal diagnosis in CLS families.     

Net nitrogen mineralization in soils under six indigenous tree species,an abandoned pasture and a secondary forest in the atlantic lowlands of costa rica

Nitrogen mineralization, nitrification potentials, pH, total N, C, extractable P and cations were measured in soils under 4-year-old, mono-specific stands of six fast-growing, native tree species, an abandoned pasture, and a 20-year-old secondary forest, as part of a study on the use of indigenous tree species for rehabilitation of soil fertility on degraded pastures at the La Selva Biological Station in the Atlantic humid lowlands of Costa Rica. Soil net nitrification potential rates were higher under two N-fixing, leguminous species,Stryphnodendron microstachyum Poepp. et Endl. (1.1–1.9 mg kg^–1 day^–1) andDalbergia tucurensis Donn. Smith (0.7–1.5 mg kg^–1 day^–1), than under the non-N-fixing trees in the plantation,Vochysia guatemalesis Don. Sm.,Vochysia ferruginea Mart,Dipteryx panamensis (Pittier) Record and Mell andHyeronima alchorneoides Fr. Allemao (0.2–0.8 mg kg^–1 day^–1). Values under the N-fixing trees were comparable to those found in secondary forest. There were no statistically significant differences in soil total N or in other nurtients between the species. Results of pH measurements done before and after incubation did not show any clear evidence of a pH drop attributable to nitrification.     

Refining the genetic map for the region flanking the X-linked hypophosphataemic rickets locus (Xp22.1–22.2)

An analysis of five of the most common cystic fibrosis (CF) mutations worldwide (F-508, R-553X, G-551D, N-1303K and G-542X) was performed in 36 Chilean patients. Polymerase chain reaction (PCR) amplification of the DNA followed by allele specific restriction enzyme analysis was used for detection. The overall frequencies of the mutations in the chromosomes analyzed were 29.2% for F-508 and 4.2% for R-553X (n=72). The G-542X, G-551D and N-1303 K mutations were absent in the Chilean sample. Our data suggest however that F-508 is not the most common CF mutation in Chilean patients. F-508 and R-553X account for only 33.4% of the alleles; 66.6% of them do not respond to the probes used and still remain uncharacterized.     

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.     

Selection of synthetic proteins to modulate the human frataxin function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a K_D value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.     

X-linked progressive mixed deafness: a new microdeletion that involves a more proximal region in Xq21.

We report a large two-generation pedigree with seven affected males segregating for an X-linked mixed conductive sensorineural deafness. The patients present with atypical Mondini-like dysplasia, dilated petrous facial canal, dilatation of the internal auditory meatus fully connected with enlarged cochlear canals, and, in one patient, a wide bulbous posterior labyrinth. Obligatory carrier females are mildly affected. Molecular characterization of this family revealed a deletion of locus DXS169, in Xq21.1. Loci DXS72 and DXS26, which, respectively, flank DXS169 proximally and distally, were intact. Since a gene responsible for X-linked progressive mixed deafness with perilymphatic gusher (DFN3) has previously been assigned by deletion mapping to a slightly more distal interval between DXS26 and DXS121, this study indicates either two different deafness genes or the involvement of a very large region in Xq21.     

Variation in trophic resources in female South American sea lions at a small geographic scale

Difference among colonies in the population structure of otariids can be driven by philopatry and/or by specializations in the foraging ecology of females. In northern Patagonia, the South American sea lion (SASL) shows some degree of spatial genetic structure among colonies from north and south zones. This study aims to explore the isotopic niche of SASL females in the last period of the pregnancy from different colonies of northern Patagonia and to consider whether the fine scale genetic spatial structuring is potentially related to variation in trophic resources. Stable isotope analysis was performed on 101 skin samples of newborn pups in 10 colonies, as a proxy for the feeding ecology of their mothers. Differences among colonies in the metrics studied revealed the plasticity of the species and support individual trophic specialization of SASL females at a small geographic scale. Also, significant differences were found in all isotopic metrics between the north and south zones. Several hypotheses were proposed to explain the differences in SASL females' isotope values (e.g., use of different foraging areas or prey, isotopic baseline variation). Nonetheless, further research is needed to better understand the relation between fine scale genetic structuring and the foraging ecology of SASL females.