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Depending on the synthesis route, the oxygen ion electrolyte Sr

Depending on the synthesis route, the oxygen ion electrolyte Sr2ScGaO5 shows two polymorphs, a brownmillerite and a cubic perovskite framework. In order to better explore oxygen diffusion pathways ...

We investigated moderate-temperature oxygen diffusion mechanisms in Sr2ScGaO5 with Brownmillerite structure type. From oxygen isotope 18O−16O exchange experiments we determined that oxygen mobility sets in above 550 °C. Temperature-dependent neutron and X-ray (synchrotron) diffraction experiments allowed us to correlate the oxygen mobility with a subtle phase transition of the orthorhombic room-temperature structure with I2mb space group toward Imma, going along with a disorder of the (GaO4)∞-tetrahedral chains. From lattice dynamical simulations we could clearly evidence that dynamic switching of the (GaO4)∞-tetrahedral chains from its R to L configuration sets in at 600 °C, thus correlating oxygen diffusion with the dynamic disorder. Oxygen ion diffusion pathways are thus constrained along the onedimensional oxygen vacancy channels, which is a different diffusion mechanism compared to that of the isostructural CaFeO2.5, where diffusion of the apical oxygen atoms into the vacant lattice sites are equally involved in the diffusion pathway. The proposed ordered room-temperature structure in I2mb is strongly supported by 17O, 45Sc, and 71Ga NMR measurements, which indicate the presence of crystallographically unique sites and the absence of local disordering effects below the phase transition. The electric field gradient tensor components measured at the nuclear sites are found to be in excellent agreement with calculated values using the WIEN2k program. The oxygen site assignment has been independently confirmed by 17O{45Sc} transfer of adiabatic populations double-resonance experiments.

International audience; Oxygen-deficient Sr$_2$ScGaO$_5$ single crystals with a cubic perovskite structure were grown by the floating-zone technique. The transparent crystals of this pure 3D oxygen electrolyte are metastable at ambient temperature, showing one-sixth of all oxygen positions vacant. While neutron single-crystal diffraction, followed by maximum entropy analysis, revealed a strong anharmonic displacements for the oxygen atoms, a predominant formation of ScO$_6$ octahedra and GaO$_4$ tetrahedra is indicated by Raman spectroscopic studies, resulting in a complex oxygen defect structure with short-range order. Temperature-dependent X-ray powder diffraction (XPD) and neutron powder diffraction (NPD) studies reveal the cubic Sr$_2$ScGaO$_5$ to be thermodynamically stable only above 1400 °C, while the stable modification below this temperature shows the brownmillerite framework with orthorhombic symmetry. Cubic Sr$_2$ScGaO$_5$ remains surprisingly kinetically stable upon heating from ambient temperature to 1300 °C, indicating a huge inertia for the retransformation toward the thermodynamically stable brownmillerite phase. Ionic conductivity investigated by impedance spectroscopy was found to be 10$^{–4}$ S/cm at 600 °C, while oxygen 18O/16O isotope exchange indicates a free oxygen mobility to set in at around 500 °C.

Oxygen-deficient Sr

Cystinosin mediates an ATP-dependent cystine efflux from lysosomes and causes, if mutated, nephropathic cystinosis, a rare inherited lysosomal storage disease. Alternative splicing of the last exon of the cystinosin sequence produces the cystinosin-LKG isoform that is characterized by a different C-terminal region causing changes in the subcellular distribution of the protein. We have constructed RFP-tagged proteins and demonstrated by site-directed mutagenesis that the carboxyl-terminal SSLKG sequence of cystinosin-LKG is an important sorting motif that is required for efficient targeting the protein to the plasma membrane, where it can mediate H+ coupled cystine transport. Deletion of the SSLKG sequence reduced cystinosin-LKG expression in the plasma membrane and cystine transport by approximately 30%, and induced significant accumulation of the protein in the Golgi apparatus and in lysosomes. Cystinosin-LKG, unlike the canonical isoform, also moves to the lysosomes by the indirect pathway, after endocytic retrieval from the plasma membrane, mainly by a clathrin-mediated endocytosis. Nevertheless, silencing of AP-2 triggers the clathrin-independent endocytosis, showing the complex adaptability of cystinosin-LKG trafficking.

Nephropathic cystinosis is multisystemic progressive disorder caused by mutations of CTNS gene that encodes for the lysosomal cystine co-transporter cystinosin, and for a less abundant isoform termed cystinosin-LKG, which is expressed in not only lysosomes but also other cell compartments. To overcome the absence of high-quality antibodies against cystinosin, we have obtained a rabbit antiserum against cystinosin-LKG and have analyzed in human tissues the expression of the two known cystinosin isoforms by RT-PCR, and the expression of cystinosin-LKG by immunohistochemistry. In most tissues, CTNS-LKG represents 5-20 % of CTNS transcripts, with the exception of the testis that expresses both isoforms in equal proportions. Cystinosin-LKG was found to be highly expressed in renal tubular cells, pancreatic islets of Langerhans, Leydig cells of the testis, mucoserous glands of the bronchial wall, melanocytes and keratinocytes. These results are parallel with many features of cystinosis, such as early onset Fanconi syndrome, male infertility, diabetes mellitus and hypopigmentation. Intermediate expression levels were of the LKG isoform observed in the gastro-intestinal tract and thyroid glands; low levels of expression were observed in the brain, skeletal and cardiac muscles.

Cell cysteine (Cys) levels and/or the [Cys/CySS] redox potential have been shown to regulate mRNA levels of the CTNS gene, which encodes for a lysosomal cystine (CySS) carrier that is defective in cystinosis. To investigate the mechanisms involved CTNS mRNA regulation, different portions of the CTNS promotor were cloned into a luciferase vector and transfected in HK2 cells. A 1.5-2.4-fold increase in luciferase activity was observed when cells were incubated in culture medium containing low CySS concentrations. Conversely, CTNS mRNA levels decreased by 47-56% in the presence of N-acetyl-L-cysteine (NAC). Chase experiments with actinomycin D (ActD) demonstrated a 3-fold stabilization of the CTNS mRNA when cells were cultured in low CySS medium for 48 h. Treatment of control cells with cyclohexamide (CHX) increased CTNS mRNA levels, suggesting that CHX blocked the synthesis of proteins involved in mRNA degradation or in repression of the CTNS gene. Finally, in vitro binding assays showed increased binding (30-110%) of the Sp-1 transcription factor to two regions of the CTNS promotor when cells were incubated in low CySS medium. These results indicate that the CTNS gene is actively regulated at the transcriptional and posttranscriptional levels and suggest that CTNS plays a pivotal role in regulating cell thiol concentrations.

The cysteine/cystine (Cys/CySS) couple represents one of the major cell thiol/disulfide systems and is involved in the regulation of several metabolic pathways and the cell redox state. Nephropathic cystinosis (NC) is an autosomal recessive disease characterized by renal cellular dysfunction due to mutations in the CTNS gene, which encodes cystinosin, a CySS lysosomal transporter. To analyze the mechanisms involved in cell damage in NC, we have investigated the effects of CTNS gene overexpression or inhibition on cell thiol/disulfide systems and vice versa. Overexpression of the CTNS gene had no remarkable effect on intracellular Cys/CySS and GSH/GSSG redox state. Silencing the CTNS gene increased cell CySS and Cys and decreased cell GSH and GSSG and increased mildly the redox state of the Cys/CySS-couple. Extracellular CySS and Cys deprivation for 48 h caused an oxidation of the Cys/CySS (73 mV) and GSH/GSSG (100 mV) redox couples and increased CTNS mRNA levels by 1.9+/-0.2-fold (p0.001). Conversely, a reduced cell environment associated with a GSH/GSSG reduction from -250.1+/-3.10 to -330.6+/-4.70 mV (p0.001) and a Cys/CySS reduction from -167.0+/-11.30 to -240.0+/-8.17 mV (p0.005) was associated with a 40% decrease in CTNS mRNA levels (p0.05). By regression analysis, CTNS gene expression was correlated with intracellular Cys level and with Cys/CySS redox state.

International audience; Metastable β-As2Te3 (R3̅m, a = 4.047 Å and c =29.492 Å at 300 K) is isostructural to layered Bi2Te3 and is known for similarly displaying good thermoelectric properties around 400 K. Crystallizing glassy-As2Te3 leads to multiphase samples, while β-As2Te3 could indeed be synthesized with good phase purity (97%) by melt quenching. As expected, β-As2Te3 reconstructively transforms into stable α-As2Te3 (C2/m, a = 14.337 Å, b = 4.015 Å, c = 9.887 Å, and β = 95.06°) at 480 K. This β → α transformation can be seen as the displacement of part of the As atoms from their As2Te3 layers into the van der Waals bonding interspace. Upon cooling, β-As2Te3 displacively transforms in two steps below TS1 = 205−210 K and TS2 = 193−197 K into a new β′ As2Te3 allotrope. These reversible and first-order phase transitions give rise to anomalies in the resistance and in the calorimetry measurements. The new monoclinic β′-As2Te3 crystal structure (P21/m, a = 6.982 Å, b = 16.187 Å, c = 10.232 Å, β = 103.46° at 20 K) was solved from Rietveld refinements of X-ray and neutron powder patterns collected at low temperatures. These analyses showed that the distortion undergone by β-As2Te3 is accompanied by a 4-fold modulation along its b axis. In agreement with our experimental results, electronic structure calculations indicate that all three structures are semiconducting with the α-phase being the most stable one and the β′-phase being more stable than the β-phase. These calculations also confirm the occurrence of a van der Waals interspace between covalently bonded As2Te3 layers in all three structures.

Estrogens have been postulated to exert a detrimental effect on spermatogenesis in vivo. Since mouse male germ cells express estrogen receptors, we have investigated whether molecular pathways are activated by estrogen stimulation of these cells. Our results demonstrate that estrogen receptor beta is expressed in mitotic and meiotic male germ cells as well as in the spermatogonia derived GC-1 cell line. By using this cell line, we show that 17-beta-estradiol triggers activation of a transcriptional response that requires a functional estrogen receptor. Moreover, GC-1 cells respond to estrogens by transiently activating a signal transduction pathway that impinges on the mitogen-activated protein kinases (MAPK) ERK1 and -2. A similar dose-dependent transient activation of ERKs was also observed in primary mouse spermatocytes in culture. Activation by the estrogen was specific because other steroids such as progesterone and dihydrotestosterone were ineffective and because it could be blocked by the selective inhibitor of the ERK pathway and by competitive inhibitors of the estrogen receptor. Finally, we observed that 17-beta-estradiol does not affect spontaneous or induced apoptosis in cultured mouse spermatocytes, indicating that the apoptotic effects observed in vivo require additional testicular components.

Spermatogonial stem cells (SSC) ensure continuous production of mammalian male gametes. In rodents, the SSC are Asingle spermatogonia (As). Gene loss and gain-of-function mutations have provided some clues into SSC function, but genetic dissection of SSC physiology has not yet been accomplished. The adaptor protein Numb is an evolutionarily conserved protein originally implicated in the control of the fate of sibling cells. Mice homozygous for deficient Numb die before embryonic day 11.5, hampering the analysis of its inactivation in postnatal male germline. Here, we have developed an experimental strategy to conditionally inactivate Numb and its homolog Numblike in the postnatal germline by in vitro delivery of cell-permeant Cre recombinase. Cre-transduced SSC isolated from wild-type mice retained their ability to self-renew and to differentiate in vivo, as shown by their ability to give rise to normal spermatogenic colonies when transplanted in recipient testes. Cre-transduced SSC from conditional mutant mouse line were able to colonize recipient testes upon transplantation. Inactivation of either Numb or Numblike in SSC did not impair the development of normal donor-derived spermatogenesis. However, compared to single-null SSC, double-null SSC generated shorter colonies in which the germ cells failed to differentiate beyond the round spermatid stage, underscoring the essential roles of both Numb and Numblike in spermatogenesis. We demonstrate the feasibility of gene inactivation in adult SSC by ex vivo Cre delivery. This provides a means to analyze the function of genes that operate on a cell-autonomous basis or those that are coupled to signals that SSC receive from bystander cells.

Contains fulltext : 69903.pdf (Publisher’s version ) (Closed access) Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [(35)S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.

BACKGROUND: Semen is the major vehicle for HIV-1 infection as it contains free and cell-associated virions and infected cells. However, the presence of HIV-1 in spermatozoa has been a matter of debate, since the sperm cell fraction may contain somatic infected cells that jeopardize the attribution of the detected virus to the spermatozoa. METHODS: Spermatozoa from 12 HIV-1 seropositive subjects were purified by multilayered Percoll gradient followed by osmotic shock. Residual presence of non-seminal cells (NCS) in purified spermatozoa, was then evaluated by cytometric and molecular analysis. HIV-1 DNA was revealed by nested PCR and in situ PCR after sperm chromatin decondensation. DNA-fragmented ejaculated spermatozoa in semen of infected subjects were detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) analysis. RESULTS: Purification procedure adopted allowed complete removal of NCS. On purified sperm cells, HIV-1 DNA was detected in 5 out of 12 subjects by nested-PCR. On crude semen of 10 out of 12 subjects, HIV -1 DNA was in situ detected in a small percentage of abnormal spermatozoa with a wide range of structural alterations. TUNEL analysis revealed an increased percentage of DNA-fragmented ejaculated spermatozoa in semen of infected subjects. CONCLUSIONS: We report molecular evidence demonstrating that HIV-1 infected subjects can ejaculate small amounts of HIV-1 DNA-positive abnormal spermatozoa. Their possible role in HIV-1 sexual transmission remains to be clarified.

Numb is an adaptor protein that is asymmetrically inherited at mitosis and controls the fate of sibling cells in different species. The role of m-Numb (mammalian Numb) as an important cell fate-determining factor has extensively been described mostly in neural tissues, particularly in progenitor cells, in the mouse. Biochemical and genetic analyses have shown that Numb acts as an inhibitor of the Notch signaling pathway, an evolutionarily conserved pathway involved in the control of cell proliferation, differentiation, and apoptosis. In the present study, we sought to determine m-Numb distribution in germ cells in the postnatal mouse testis. We show that all four m-Numb isoforms are widely expressed during postnatal testis development. By reverse transcriptase-PCR and western blot analyses, we further identify p71 as the predominantly expressed isoform in germ cells. Moreover, we demonstrate through co-immunoprecipitation studies that m-Numb physically associates with Ap2a1, a component of the endocytotic clathrin-coated vesicles. Finally, we employed confocal immunofluorescence microscopy of whole mount seminiferous tubules and isolated germ cells to gain more insight into the subcellular localization of m-Numb. These morphological analyses confirmed m-Numb and Ap2a1 co-localization. However, we did not observe asymmetric localization of m-Numb neither in mitotic spermatogonial stem cells nor in more differentiated spermatogonial cells, suggesting that spermatogonial stem cell fate in the mouse does not rely on asymmetric partitioning of m-Numb.

Estrogens have been postulated to exert a detrimental effect on spermatogenesis in vivo. Since mouse male germ cells express estrogen receptors, we have investigated whether molecular pathways are activated by estrogen stimulation of these cells. Our results demonstrate that estrogen receptor beta is expressed in mitotic and meiotic male germ cells as well as in the spermatogonia derived GC-1 cell line. By using this cell line, we show that 17-beta-estradiol triggers activation of a transcriptional response that requires a functional estrogen receptor. Moreover, GC-1 cells respond to estrogens by transiently activating a signal transduction pathway that impinges on the mitogen-activated protein kinases (MAPK) ERK1 and -2. A similar dose-dependent transient activation of ERKs was also observed in primary mouse spermatocytes in culture. Activation by the estrogen was specific because other steroids such as progesterone and dihydrotestosterone were ineffective and because it could be blocked by the selective inhibitor of the ERK pathway and by competitive inhibitors of the estrogen receptor. Finally, we observed that 17-beta-estradiol does not affect spontaneous or induced apoptosis in cultured mouse spermatocytes, indicating that the apoptotic effects observed in vivo require additional testicular components.

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self-maintain and to produce a transit-amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil-sensitive Hoechst dye-effluxing properties, which define the characteristic "side population" (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a "side-population" (T-SP), which is Sca-1pos, Ep-CAMpos, EE2 pos, alpha6-integrin pos, and alpha(v)-integrin neg. A 13-fold enrichment in SSC activity was observed when sorted T-SP cells from ROSA 26 mice were transplanted in busulfan-treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T-SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.