Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-recessive spastic paraplegia, including Kjellin syndrome

Hereditary spastic paraplegias (HSPs) are genetically and phenotypically heterogeneous disorders. Both "uncomplicated" and "complicated" forms have been described with various modes of inheritance. Sixteen loci for autosomal-recessive "complicated" HSP have been mapped. The SPG15 locus was first reported to account for a rare form of spastic paraplegia variably associated with mental impairment, pigmented maculopathy, dysarthria, cerebellar signs, and distal amyotrophy, sometimes designated as Kjellin syndrome. Here, we report the refinement of SPG15 to a 2.64 Mb genetic interval on chromosome 14q23.3-q24.2 and the identification of ZFYVE26, which encodes a zinc-finger protein with a FYVE domain that we named spastizin, as the cause of SPG15. Six different truncating mutations were found to segregate with the disease in eight families with a phenotype that included variable clinical features of Kjellin syndrome. ZFYVE26 mRNA was widely distributed in human tissues, as well as in rat embryos, suggesting a possible role of this gene during embryonic development. In the adult rodent brain, its expression profile closely resembled that of SPG11, another gene responsible for complicated HSP. In cultured cells, spastizin colocalized partially with markers of endoplasmic reticulum and endosomes, suggesting a role in intracellular trafficking.     

In Situ Fe and S isotope analyses in pyrite from the 3.2 Ga Mendon Formation (Barberton Greenstone Belt,South Africa): Evidence for early microbial iron reduction

On the basis of phylogenetic studies and laboratory cultures, it has been proposed that the ability of microbes to metabolize iron has emerged prior to the Archaea/Bacteria split. However, no unambiguous geochemical data supporting this claim have been put forward in rocks older than 2.7–2.5 giga years (Gyr). In the present work, we report in situ Fe and S isotope composition of pyrite from 3.28‐ to 3.26‐Gyr‐old cherts from the upper Mendon Formation, South Africa. We identified three populations of microscopic pyrites showing a wide range of Fe isotope compositions, which cluster around two δ⁵⁶Fe values of ?1.8‰ and +1‰. These three pyrite groups can also be distinguished based on the pyrite crystallinity and the S isotope mass‐independent signatures. One pyrite group displays poorly crystallized pyrite minerals with positive Δ³³S values > +3‰, while the other groups display more variable and closer to 0‰ Δ³³S values with recrystallized pyrite rims. It is worth to note that all the pyrite groups display positive Δ³³S values in the pyrite core and similar trace element compositions. We therefore suggest that two of the pyrite groups have experienced late fluid circulations that have led to partial recrystallization and dilution of S isotope mass‐independent signature but not modification of the Fe isotope record. Considering the mineralogy and geochemistry of the pyrites and associated organic material, we conclude that this iron isotope systematic derives from microbial respiration of iron oxides during early diagenesis. Our data extend the geological record of dissimilatory iron reduction (DIR) back more than 560 million years (Myr) and confirm that micro‐organisms closely related to the last common ancestor had the ability to reduce Fe(III).     

Microsatellite null alleles and estimation of population differentiation

Microsatellite null alleles are commonly encountered in population genetics studies, yet little is known about their impact on the estimation of population differentiation. Computer simulations based on the coalescent were used to investigate the evolutionary dynamics of null alleles, their impact on F(ST) and genetic distances, and the efficiency of estimators of null allele frequency. Further, we explored how the existing method for correcting genotype data for null alleles performed in estimating F(ST) and genetic distances, and we compared this method with a new method proposed here (for F(ST) only). Null alleles were likely to be encountered in populations with a large effective size, with an unusually high mutation rate in the flanking regions, and that have diverged from the population from which the cloned allele state was drawn and the primers designed. When populations were significantly differentiated, F(ST) and genetic distances were overestimated in the presence of null alleles. Frequency of null alleles was estimated precisely with the algorithm presented in Dempster et al. (1977). The conventional method for correcting genotype data for null alleles did not provide an accurate estimate of F(ST) and genetic distances. However, the use of the genetic distance of Cavalli-Sforza and Edwards (1967) corrected by the conventional method gave better estimates than those obtained without correction. F(ST) estimation from corrected genotype frequencies performed well when restricted to visible allele sizes. Both the proposed method and the traditional correction method have been implemented in a program that is available free of charge at http://www.montpellier.inra.fr/URLB/. We used 2 published microsatellite data sets based on original and redesigned pairs of primers to empirically confirm our simulation results.     

Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis

The catalytic adenyl cyclase (AC) domain of the protein CyaA from Bordetella pertussis is activated by interaction with the C terminal lobe of calmodulin (C-CaM). The AC/C-CaM complex displays an elongated shape, but hydrodynamics measurements on the isolated AC domain allowed to characterize the shape of the protein as spherical. Here, we study by molecular dynamics simulations the complexes between AC and the apo and Ca(2+)-loaded C-CaM, as well as the isolated AC, to characterize the features of AC conformational variability and of AC/C-CaM interaction. The removal of calcium ions from C-CaM increases the AC flexibility, but the removal of C-CaM induces a dramatic drift of the AC conformation. Isolated AC conformations show a general tendency to become less elongated, as the two protein extremities (regions SA and CB) tend to get closer. An analysis of the energetic influences between the C-CaM and the AC regions shows a simple influence scheme, in agreement with the high affinity of AC to CaM. In this scheme, a single influence is observed from C-CaM to the region CA of the AC domain. This influence is correlated to the presence of hydrogen bonds involving residues from C-CaM, and from regions CA, C-terminal tail, and catalytic loop of AC. This study reveals a C-CaM/AC interaction picture where C-CaM stabilizes AC by a steric hindrance on the conformational drift of SA, whereas the Ca(2+) ions allow further stabilization by the establishment of a hydrogen bond network extending from C-CaM to the AC catalytic loop.     

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy

Objective

There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials and Methods

pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results

After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion

This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.     

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.     

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.