Femtosecond phase-transition in hard x-ray excited bismuth.

The evolution of bismuth crystal structure upon excitation of its A1g phonon has been intensely studied with short pulse optical lasers. Here we present the first-time observation of a hard x-ray induced ultrafast phase transition in a bismuth single crystal at high intensities (~1014 W/cm2). The lattice evolution was followed using a recently demonstrated x-ray single-shot probing setup. The time evolution of the (111) Bragg peak intensity showed strong dependence on the excitation fluence. After exposure to a sufficiently intense x-ray pulse, the peak intensity dropped to zero within 300 fs, i.e. faster than one oscillation period of the A1g mode at room temperature. Our analysis indicates a nonthermal origin of a lattice disordering process, and excludes interpretations based on electron-ion equilibration process, or on thermodynamic heating process leading to plasma formation.

OMNY-A tOMography Nano crYo stage.

For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved via dedicated laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy. Cryogenic sample conditions are maintained via conductive cooling. 90 K can be reached when using liquid nitrogen as coolant, and 10 K is possible with liquid helium. A cryogenic sample-change mechanism permits measurements of cryogenically fixed specimens. We compare images obtained with OMNY with older measurements performed using a nitrogen gas cryo-jet of stained, epoxy-embedded retina tissue and of frozen-hydrated Chlamydomonas cells.

Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors.

The glycoprotein hormone receptors (thyrotrophin receptor, TSHr; luteinizing hormone/chorionic gonadotrophin receptor, LH/CGr; follicle-stimulating hormone receptor, FSHr) constitute a subfamily of rhodopsin-like G protein-coupled receptors (GPCRs) with a long N-terminal extracellular extension responsible for high-affinity hormone binding. These ectodomains contain two cysteine clusters flanking nine leucine-rich repeats (LRR), a motif found in several protein families involved in protein-protein interactions. Similar to the situation described recently in CCR5, we demonstrate here that the TSHr, as it is present at the cell surface, is sulfated on tyrosines in a motif located downstream of the C-terminal cysteine cluster. Sulfation of one of the two tyrosines in the motif is mandatory for high-affinity binding of TSH and activation of the receptor. Site-directed mutagenesis experiments indicate that the motif, which is conserved in all members of the glycoprotein hormone receptor family, seems to play a similar role in the LH/CG and FSH receptors.

Purification and characterization of a soluble bioactive amino-terminal extracellular domain of the human thyrotropin receptor.

The amino-terminal ectodomain of the human TSH receptor has been expressed at the surface of CHO cells as a glycosylphosphatidylinositol-anchored molecule containing a 10-residue histidine tag close to its C terminus. The soluble ectodomain could be released from the cells by treatment with a glycosylphosphatidylinositol-phospholipase C and purified to apparent homogeneity by cobalt-Sepharose chromatography. Two nanomoles of material was obtained, which was suitable for analysis by mass spectrometry. This allowed the identification of four out of the six potential N-glycosylation sites as being effectively glycosylated. A proportion of the purified soluble ectodomain displayed specific binding of (125)I-labeled TSH, allowing for the first time performance of classical saturation binding experiments. Two classes of high-affinity binding sites were identified: site A, K(d) 0.014 nM; site B, K(d) 0.83 nM. The significance of site A, whose affinity is much higher than for the holoreceptor at the surface of intact cells, remains to be clarified. The purified ectodomain was capable of inhibiting efficiently the thyroid stimulating activity of immunoglobulins from patients with Graves' disease. It allowed computation of the amounts of these immunoglobulins in patient's serum, giving values up to 10 microg/mL. Contrary to all currently available assays, the soluble ectodomain of the TSH receptor purified in a functionally competent conformation allows direct studies of its interactions with TSH and autoantibodies and opens the way to structural studies.

Human thyroperoxidase in its alternatively spliced form (TPO2) is enzymatically inactive and exhibits changes in intracellular processing and trafficking.

Thyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and the alternatively spliced isoform TPO2. Pulse-chase studies showed that TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity of TPO2 to endo-beta-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannose-type structures. Cell surface biotinylation experiments showed that the two isoforms also differ in their intracellular trafficking. TPO2 was totally retained in the cell, whereas 15% of TPO1 reached the cell surface. The inability of TPO2 to come out of the intracellular compartments was related to structural changes in the molecule. Evidence of these changes was obtained through the lack of recognition of TPO2 by half of the 13 TPO monoclonal antibodies tested in immunoprecipitation experiments. Our data suggest that because of an improper folding, TPO2 is trapped in the endoplasmic reticulum and rapidly degraded. The failure of incorporation of [14C]aminolevulinic acid in the cultured cells showed that TPO2 did not bind to heme, whereas TPO1 did. This result was confirmed through a guaiacol assay showing that TPO2 is enzymatically inactive.

Biosynthesis and metabolism of 2-iodohexadecanal in cultured dog thyroid cells.

2-Iodohexadecanal (2-IHDA) is a major thyroid iodolipid. It mimics the main regulatory effects of iodide on thyroid metabolism: inhibition of H2O2 production and of adenylyl cyclase. The biosynthesis of 2-IHDA and its metabolism have been investigated in cultured dog thyroid cells maintained in a differentiated state by forskolin. Incubation of these cells with [9,10-3H]hexadecan-1-ol or [9,10-3H]palmitic acid labeled several phospholipids, but [9, 10-3H]hexadecan-1-ol was selectively incorporated into plasmenylethanolamine. In the presence of an exogenous H2O2 generating system (glucose oxidase), iodide induced the production of [9,10-3H]2-IHDA from [9,10-3H]hexadecan-1-ol-labeled cells but not from [9,10-3H]palmitic acid-labeled cells. 2-IHDA was also generated during the lactoperoxidase-catalyzed iodination of brain and heart plasmalogens, and of ethyl hexadec-1-enyl ether, a synthetic vinyl ether-containing compound. Taken together, these results show that thyroid 2-IHDA is derived from plasmenylethanolamine via an attack of reactive iodine on the vinyl ether group. 2-Iodohexadecan-1-ol (2-IHDO) was also detected in these studies; it was formed later than 2-IHDA, and thyroid cells converted exogenous 2-IHDA into 2-IHDO in a time-dependent way. The ratio of 2-IHDO/2-IHDA increased with H2O2 production and decreased as a function of iodide concentration. An aldehyde-reducing activity was detected in subcellular fractions of the horse thyroid. No formation of 2-iodohexadecanoic acid could be detected. Reduction into the biologically inactive 2-IHDO is thus a major metabolic pathway of 2-IHDA in dog thyrocytes.

The thyroperoxidase doublet is not produced by alternative splicing.

Thyroperoxidase is a membrane-bound, heme-containing enzyme which catalyses iodination of thyroglobulin and coupling of resulting iodotyrosines to produce thyroid hormone. In addition to the full length molecule of 933 amino acids (TPO1), Northern blotting and sequencing have revealed several shorter transcripts. The most abundant is a species lacking 171 nucleotides in which the alternative splicing results in the deletion of codons 533-590 in exon 10 (TPO2). Evidence for TPO2 transcripts being translated into a protein is lacking, but in Western blots TPO invariably appears as a doublet of 110 and 105 kDa. In the present study we have produced two recombinant fusion proteins for: (i) the 57 amino acids which are spliced out in TPO2 and (ii) for the 20 amino acids which bridge the splice site (10 amino acids on both sides). Both recombinant fragments have been produced in the pMAL-cRI vector as a maltose-binding protein (MBP) fusion, permitting their purification from a bacterial lysate on an amylose column. Rabbits have been immunized by intradermal injection of 500 micrograms of fusion protein, initially in complete Freund's adjuvant followed by two boosts, at 2-week intervals, in incomplete Freund's adjuvant. The resulting high titre immune sera (IS) were reactive with the relevant immunising antigens, when tested by ELISA. Depletion of each serum by passage through an MBP-CNBr Sepharose column allowed purification of antibodies against the relevant peptides, as demonstrated by ELISA with the appropriate fusion protein and MBP. This demonstrates that we have produced specific polyclonal antibodies for the 57 amino acids unique to TPO1 and for the amino acid segment bridging the splice site, found in TPO2. These polyclonal antibodies were used in Western blotting experiments with normal and Graves' thyroid membranes, in reducing and non-reducing conditions. Monoclonal 47/C21 which recognises a linear epitope (amino acids residues 710-722) common to TPO1 and TPO2 was used as a control. In non-reducing conditions, we observed a broad signal at 105-110 kDa, which appeared to comprise two bands, with both polyclonal antibodies and the monoclonal. There was no difference in the image between the normal and the Graves' thyroid. In reducing conditions, the broad signal resolved clearly into two distinct bands, one at 105 and the other at 110 kDa. Once again we observed exactly the same pattern of reactivity with all three antibodies both in normal and Graves' glands. We conclude that the TPO doublet is not the consequence of translation of TPO2.

Inhibition of human thyroid adenylyl cyclase by 2-iodoaldehydes.

2-Iodohexadecanal (IHDA), which can be formed upon addition of iodine to the vinyl ether group of plasmalogens, has been identified as a major thyroid iodolipid (Pereira et al. (1990) J. Biol. Chem. 265, 17018-17025). In this study, we have investigated the possibility that it would be a mediator of the inhibitory effect of iodide on thyroid adenylyl cyclase. In human thyroid membranes, IHDA inhibited the adenylyl cyclase activity stimulated by thyrotropin (TSH), GTP-gamma-S or forskolin (FSK), whereas it did not decrease the specific binding of TSH to its receptors. The inhibitory effect on the cyclase reached a maximum after a 1-h-pre-incubation of the membranes with IHDA at 30 degrees C and was poorly reversible. It was also observed following a 4-h incubation with IHDA at 4 degrees C, a condition in which adenylyl cyclase is protected against heat inactivation. IHDA decreased the Vmax of adenylyl cyclase, but had no effect on the Km for ATPMg2-.IHDA also inhibited the FSK-stimulated adenylyl cyclase activity in liver and kidney cortex membranes, but had no effect on the Mg(2+)-ATPase activity of thyroid membranes. The inhibitory effect of IHDA has also been demonstrated in intact cells. As in membranes, IHDA decreased the rise in cAMP induced by TSH in cultured dog thyroid cells and this inhibition was maintained following pretreatment of the cells with pertussis toxin. In order to evaluate the specificity of the IHDA action, various analogs have been synthesized. This study has permitted the identification of two major structural features required for the inhibition of human thyroid adenylyl cyclase; the terminal aldehyde function and an iodine atom at C2, other halogens being ineffective. In conclusion, we have shown that IHDA exerts a direct inhibitory effect at or near adenylyl cyclase; all the properties of this effect characterized so far are identical to those of the adenylyl cyclase inhibition obtained following the exposure of thyroid tissue to iodide.

Inhibition of H2O2 production by iodoaldehydes in cultured dog thyroid cells.

2-Iodohexadecanal (IHDA) has been identified as a major thyroid iodolipid which can be formed upon addition of iodine to the vinyl ether group of plasmalogens (Pereira et al., 1990). In order to test whether IHDA plays a role in the thyroid autoregulation by iodide, we have investigated its effects on the production of H2O2 by cultured dog thyroid cells. IHDA inhibited the formation of H2O2 in dog thyroid cells stimulated by carbamylcholine (CCHOL). In the presence of BSA, which potentiated its action, the effect of IHDA was maximal after 2 h and had an IC50 around 5 microM. The effect of IHDA was not decreased by methimazole, which abolished the inhibition by iodide. IHDA also inhibited the stimulatory effect of bradykinin, but had only a marginal effect on the production of H2O2 induced by ionomycin or phorbol 12-myristate 13-acetate (PMA). The accumulation of inositol phosphates in CCHOL-stimulated thyroid cells was decreased by IHDA. As evaluated by measurements of 51Cr release and [3H]thymidine incorporation into DNA, IHDA had no adverse effect on thyroid cell viability. Several analogs of IHDA, of which the synthesis is described, have been tested for their inhibitory activity. This allowed the identification of two major structural features required for the biological activity: the carbonyl group at C1 and an halogen atom at C2, with iodine conferring a greater activity than bromine, while chlorine and fluorine were inactive. In conclusion, IHDA inhibits the production of H2O2 in CCHOL-stimulated dog thyroid cells by decreasing the phospholipase C cascade activity. This effect involves both the aldehyde function and the iodine atom. These results suggest that IHDA might be the mediator of some of the regulatory actions of iodide on the thyroid gland.