Search for Active-Sterile Antineutrino Mixing Using Neutral-Current Interactions with the NOvA Experiment.

This Letter reports results from the first long-baseline search for sterile antineutrinos mixing in an accelerator-based antineutrino-dominated beam. The rate of neutral-current interactions in the two NOvA detectors, at distances of 1 and 810 km from the beam source, is analyzed using an exposure of 12.51×10^{20} protons-on-target from the NuMI beam at Fermilab running in antineutrino mode. A total of 121 of neutral-current candidates are observed at the far detector, compared to a prediction of 122±11(stat.)±15(syst.) assuming mixing only between three active flavors. No evidence for ν[over ¯]_{µ}→ν[over ¯]_{s} oscillation is observed. Interpreting this result within a 3+1 model, constraints are placed on the mixing angles θ_{24}<25° and θ_{34}<32° at the 90% C.L. for 0.05 eV^{2}≤Δm_{41}^{2}≤0.5 eV^{2}, the range of mass splittings that produces no significant oscillations at the near detector. These are the first 3+1 confidence limits set using long-baseline accelerator antineutrinos.

Increase in polysialyltransferase gene expression following LTP in adult rat dentate gyrus.

Neural cell adhesion molecule (NCAM) is frequently associated with polysialic acid (PSA), and its function is highly dependent on this polysialylation. PSA-NCAM plays an important role in synaptic plasticity in the hippocampus. STX and PST are the enzymes responsible for NCAM polysialylation. We investigated whether unilateral long-term potentiation (LTP) induction in vivo, in adult rat dentate gyrus (DG), triggered NCAM polysialylation by STX and PST produced in the hippocampus. We found that levels of STX and PST mRNA increased strongly since the early stage of hippocampal LTP and remained high during the maintenance of DG-LTP for 4 h. This rapid increase in polysialyltransferase gene expression occurred in both the hippocampi, probably resulting from bilateral LTP induction by strong unilateral HFS. Thus, LTP triggers interhemispheric molecular changes in the hippocampal network. This study is the first to describe the effects of LTP induction and maintenance on polysialyl-transferases in vivo. Our findings suggest that hippocampal synaptic remodeling requires NCAM polysialylation.

Dentists of tomorrow 2020: An analysis of the results of the 2020 ADEA Survey of U.S. Dental School Seniors.

PURPOSE/OBJECTIVES: This study examines the journey of U.S. dental schools' predoctoral senior class of 2020, from the influences on and their motivations to pursue careers in dentistry, aspects of their dental school experiences, to plans upon graduation and the investment in their careers. METHODS: The study is an analysis of the results of the ADEA Survey of Dental School Seniors, 2020 Graduating Class. Each year, ADEA surveys senior predoctoral students from the accredited U.S. dental schools. Whenever feasible, the answers of the survey respondents from the 2020 class were compared with their 2015 counterparts. RESULTS: The analysis revealed that 46% of the 2020 respondents decided to become a dentist before going to undergraduate college, more than the proportion of those deciding while in college (42%). When it comes to preparedness to practice dentistry, the responses indicated a high level of readiness to go into the profession. Seventy-seven percent of survey participants reported the COVID-19 pandemic did not affect their plans after graduation. Between 2015 and 2020, the share of survey respondents who planned to go into advanced dental education immediately after graduation increased from 35% to 40%. Almost a third of the 2020 respondents who planned to go into private practice immediately upon graduation intended to join a Dental Service Organization (DSO). Grants and scholarships represented a higher share of the average funding for dental education for the 2020 respondents than five years ago. The share of respondents expecting to graduate without any loans to finance their dental degrees and predental education (educational debt) increased significantly, from 12% in 2015 to 17% in 2020. CONCLUSION(S): This research shows that during these uncertain times, U.S. dental schools continued their mission to train and graduate oral health professionals fully prepared to go into the profession.

[Vertebroplasty: 10 years clinical and radiological follow-up]. / Vertébroplastie: évaluation clinique et radiologique à plus de dix ans.

OBJECTIVES: Ten years follow-up of the first patients treated with percutaneous vertebroplasty. PATIENT AND METHODS: Eighteen patients were retrospectively reviewed having undergone vertebroplasty in our centre between 1989 and 1998. Eight were treated for angioma, eight for osteoporotic compression and two followed for myeloma. They all underwent clinical and radiological evaluation in 2007 (standard X-rays, CT scan and MRI). These examinations were compared to prior baseline pre- and post-therapeutic images. RESULTS: Radiological characteristic of cement remained unchanged in the long term and there was no modification of anatomical structures in contact with it. Even if the distribution of cement was asymmetrical there was no fracture of the treated vertebras at distance. Degenerative changes of discs facing the vertebroplasty were not more pronounced than for distant discs. We found no significant signal or density anomaly of disc in contact direct with cement. 38.8 % of the patients presented new fractures (n=30). Seventy percent of the fractures were multiple and contiguous. In the long term, all patients reported improvement of pain after the procedure. CONCLUSION: In our series, we found a good stability of treatment over time. This study shows the long-term safety of percutaneous acrylic vertebroplasty, in particular harmlessness of cement for bone and discs in contact.

Masking technique for coating thickness control on large and strongly curved aspherical optics.

We discuss a method to control the coating thickness deposited onto large and strongly curved optics by ion beam sputtering. The technique uses an original design of the mask used to screen part of the sputtered materials. A first multielement mask is calculated from the measured two-dimensional coating thickness distribution. Then, by means of an iterative process, the final mask is designed. By using such a technique, it has been possible to deposit layers of tantalum pentoxide having a high thickness gradient onto a curved substrate 500 mm in diameter. Residual errors in the coating thickness profile are below 0.7%.

[Bithalamic infarct: is there an evocative aspect? Radioclinical study]. / Accident vasculaire ischémique bithalamique: existe-t-il un tableau évocateur ? Etude radioclinique.

INTRODUCTION: Bithalamic paramedian infarcts are uncommon. This stroke results in a complex clinical syndrome. CASE REPORT: We report four cases of bithalamic paramedian infarcts with a presumed mechanism of occlusion of a single thalamic paramedian artery. DISCUSSION: This normal anatomic variant corresponds to an asymmetrical common trunk for the two thalamosubthalamic paramedian arteries arising from a P1 segment (type IIb in the G. Percheron classification dating from 1977). A literature analysis (from 1985 to 2006) allowed us to identify the most widely reported clinical signs. Four main clinical findings are described: vertical gaze palsy (65%), memory impairment (58%), confusion (53%) and coma (42%). We also found these symptoms in our patients but rarely associated; however, all four patients had exhibited episodes of drowsiness. In this article, we discuss the anatomy-function correlation responsible for such clinical variability. CONCLUSION: Clinicians should be aware of this diagnosis to better understand the imaging results which provide confirmation. Although the literature describes frequently severe consciousness disorders such as coma, this diagnosis must also be considered in patients presenting a simple fluctuation of consciousness, e.g. hypersomnia.

[Arterial spin labeling: state of the art]. / Etat de l'art sur le Marquage de Spin Artériel ou ASL.

UNLABELLED: Arterial spin labeling (ASL) perfusion MR imaging is a technique by which water from circulating arterial blood is magnetically labeled and acts as a diffusible tracer allowing non-invasive measurement of cerebral blood flow. In this paper, the technique and current applications in neuroimaging will be reviewed. CURRENT STATUS: First, the technical principles of ASL will be reviewed and both available techniques (continuous and pulsed ASL) explained. A review of the literature will demonstrate advances with the techniques of ASL and its clinical impact. Clinical research involves normal volunteers and patients with ischemic and tumoral pathologies. CONCLUSION: Recent technical advances have improved the sensitivity of ASL perfusion MR imaging. The routine clinical use of ASL at 3.0 Tesla should increase over the next few years.

Role of selenium addition to CdZnTe matrix for room-temperature radiation detector applications.

Because of its ideal band gap, high density and high electron mobility-lifetime product, cadmium zinc telluride (CdZnTe or CZT) is currently the best room-temperature compound-semiconductor X- and gamma-ray detector material. However, because of its innate poor thermo-physical properties and above unity segregation coefficient for Zn, the wide spread deployment of this material in large-volume CZT detectors is still limited by the high production cost. The underlying reason for the low yield of high-quality material is that CZT suffers from three major detrimental defects: compositional inhomogeneity, high concentrations of dislocation walls/sub-grain boundary networks and high concentrations of Te inclusions/precipitates. To mitigate all these disadvantages, we report for the first time the effects of the addition of selenium to the CZT matrix. The addition of Se was found to be very effective in arresting the formation of sub-grain boundaries and its networks, significantly reducing Zn segregation, improving compositional homogeneity and resulting in much lower concentrations of Te inclusions/precipitates. Growth of the new quaternary crystal Cd1-xZnxTe1-ySey (CZTS) by the Traveling Heater Method (THM) is reported in this paper. We have demonstrated the production of much higher yield according to its compositional homogeneity, with substantially lower sub-grain boundaries and their network, and a lower concentration of Te inclusions/precipitates.

Efficient and tolerant resonant grating coupler for multimode optical interconnections.

More than 60% overall coupling efficiency is achieved in the demonstrator of an optical interconnect comprising an input grating coupler, a multimode slab waveguide section and an output grating coupler. The grating coupling strength is enhanced by means of a leaky mode resonance. The efficiency of the resonant grating coupler compares favourably with the performancs reported on mirror inserts.

Morphological and functional imaging of neck paragangliomas.

OBJECTIVE: To review the optimal techniques for localization and characterization of neck paragangliomas (PGL). MATERIAL AND METHODS: Systematic review of the literature from the PubMed/Medline database. RESULTS: Neck PGL are hypervascular tumours essentially arising from paraganglionic tissue situated at the carotid bifurcation (carotid body) and along the vagus nerve. Morphological and functional imaging are indicated to confirm the diagnosis, identify multifocal disease and for local and regional staging. MR angiography is the noninvasive technique of choice. CT scan and especially CT angiography are excellent alternatives for diagnosis and staging. Conventional arteriography remains useful preoperatively for embolization and occlusion tests. Functional imaging allows localization and characterization of PGLs. Somatostatin receptor scintigraphy (SRS) was the reference imaging technique for staging of sporadic PGLs. The indications for PET imaging have been extended over recent years in parallel with the development of new tracers such as [18F]-FDOPA PET or 68Gallium-labelled DOTA peptides. 68Gallium-labelled DOTA peptides has become the first-line imaging modality in the evaluation of cervical PGLs, regardless of the genetic background. CONCLUSION: Morphological and functional imaging is essential for the staging of neck PGL.

Pituitary transcription factors: from congenital deficiencies to gene therapy.

Despite the existence of interspecies phenotypic variability, animal models have yielded valuable insights into human pituitary diseases. Studies on Snell and Jackson mice known to have growth hormone, prolactin and thyroid-stimulating hormone deficiencies involving the hypoplastic pituitary gland have led to identifying alterations of the pituitary specific POU homeodomain Pit-1 transcription factor gene. The human phenotype associated with rare mutations in this gene was found to be similar to that of these mice mutants. Terminal differentiation of lactotroph cells and direct regulation of the prolactin gene both require interactions between Pit-1 and cell type specific partners, including panpituitary transcriptional regulators such as Pitx1 and Pitx2. Synergistic activation of the prolactin promoter by Pitx factors and Pit-1 is involved not only in basal condition, but also in responsiveness to forskolin, thyrotrophin-releasing-hormone and epidermal growth factor. In corticotroph cells, Pitx1 interacts with Tpit. Tpit mutations have turned out to be the main molecular cause of neonatal isolated adrenocorticotrophin deficiency. This finding supports the idea that Tpit plays an essential role in the differentiation of the pro-opiomelanocortin pituitary lineage. The effects of Pit-1 are not restricted to hormone gene regulation because this factor also contributes to cell division and protects the cell from programmed cell death. Lentiviral vectors expressing a Pit-1 dominant negative mutant induced time- and dose-dependent cell death in somatotroph and lactotroph adenomas in vitro. Gene transfer by lentiviral vectors should provide a promising step towards developing an efficient specific therapeutic approach by which a gene therapy programme for treating human pituitary adenomas could be based.

Phosphatidylcholine requirement for the N-glycosylation of synthetic peptides by detergent-solubilized oligosaccharyltransferase.

The ability of dolichyl-P-P-oligosaccharide:peptide oligosaccharyltransferase to use exogenous substrates (a previously labeled oligosaccharide lipid and an Asn-X-Thr containing heptapeptide) is shown to require phospholipid. The enzyme was extracted from porcine thyroid rough microsomes using NaCl-Nonidet P-40. When measured at low concentration, in a neutral detergent-containing medium, it undergoes a rapid loss of activity, which renders impossible quantitative estimates in the range of 0-50 micrograms microsomal protein/50 microliters assay. We observed that inactivation could be prevented by supplementing the assay with a previously heat-treated suspension of microsomes in neutral detergent, or with the corresponding extract. Further investigation revealed that phospholipids are responsible for this enzyme stabilization, since phospholipase A2 and phospholipase C treatments were both able to abolish this effect. When individual phospholipids were compared for their protective efficiency, egg yolk phosphatidylcholine was found to be by far the most efficient. Phosphatidylglycerol, phosphatidylinositol and phosphatidylserine were only slightly effective, while phosphatidylethanolamine and lysophosphatidylcholine had no effect at all. Of those tested, partly unsaturated phosphatidylcholines with 16-18 carbon atom acyl chains were the most active, at an optimal concentration of 1-2 mM. Under these conditions a Km of 15 microM was measured for the acceptor, a synthetic ribonuclease heptapeptide, and a Km of 0.55 microM for the donor, dolichyl-P-P-GlcNAc2-Man9-Glc2-3. These findings were confirmed by subjecting a sodium deoxycholate extract to depletion of endogenous lipids by gel filtration. Enzyme activity was totally abolished and then restored (up to now only partially) by addition of phosphatidylcholine.

Massa mediastinal anterior em jovem: aneurisma de artéria descendente anterior como diagnóstico diferencial, um relato de caso / Anterior mediastinal mass in a young woman: anterior descending artery aneurysm as a differential diagnosis, a case report

INTRODUÇÃO: As massas mediastinais anteriores (MMA) são as mais comuns do grupo de tumores do mediastino. O timoma é a forma mais comum, já os tumores de células germinativas mediastinais, os quais caracterizam um grupo heterogêneo de neoplasias, correspondem a cerca de 15%, dentre eles o teratoma. O presente caso, no entanto, elucida a importância do diagnóstico diferencial para essas MMAs. MÉTODOS: Relato de caso com análise de prontuário. RESULTADOS: Masculino, 14 anos, antecedentes patológicos de pseudoaneurisma de artéria mamária interna esquerda embolizado aos 8 anos e aftas orais recorrentes. Relatou dor anginosa há 5 meses de média intensidade sem irradiação, com melhora à analgesia simples e repouso. Procurou atendimento médico devido à piora dos sintomas há um mês, acompanhados de tosse seca, dispneia e febre. Admitido com eletrocardiograma normal e hipótese diagnóstica de pneumonia complicada com derrame pleural a esquerda, o paciente realizou ecocardiograma transtorácico (ECOTT) com evidência de derrame pericárdico leve. Em uso de antibioticoterapia com pesquisa infecciosa negativa realizou angiotomografia de tórax, a qual evidenciou tromboembolismo pulmonar à direita e massa na região apical do ventrículo direito (VD) de 3,4cm. Encaminhado a um hospital terciário para investigação realizou novo ECOTT: imagem heterogênea na porção médio-apical do VD, medindo 5,1 cm x 4,2 cm, podendo corresponder a trombo ou massa. Seguiu investigação com ressonância nuclear magnética cardíaca que mostrou massa intramiocárdica, sendo aventado hipótese de teratoma. Heart Team optou por prosseguir investigação através de angiotomografia de coronárias, a qual mostrou em artéria descendente anterior (ADA) aneurisma sacular em terço distal medindo 63x53mm. A cinecoronarioangiografia subsequente evidenciou ADA ocluída no terço médio com aspecto de compressão extrínseca. Equipe manteve tratamento conservador e investigação diagnóstica etiológica em conjunto com reumatologia, cuja suspeita firmou-se em Doença de Behçet (DB), desordem inflamatóri multisistêmica. CONCLUSÃO: O caso em questão partiu de investigação de MMAs com análise de imagens criteriosa, a qual evidenciou aneurisma de ADA, com hipótese no contexto de DB, cujo acometimento de coronárias é raro. Dessa maneira, houve impacto profundo na opção terapêutica. Dessa forma demostramos a importância do estudo de imagem detalhado para diagnóstico de massas cardíacas. O paciente do caso recebeu pulsoterapia com corticoesteróide e seguiu em acompanhamento com reumatologia e cardiologia de hospital terciário.

Effects of tunicamycin and N-linked oligosaccharide-processing inhibitors on the morphology of cultured porcine thyroid cells.

The effects of tunicamycin and of N-linked oligosaccharide-processing inhibitors on the ability of cultured porcine thyroid cells to adhere to a plastic support and to form organized structures were examined. The culture conditions used allowed the epithelial cells to adhere to the support and to form either a monolayer (no thyrotropin) or follicles (thyrotropin 4 mU/ml). The follicles thus obtained tend to disappear after 8 to 9 days, giving rise to a monolayer. Tunicamycin prevented both cell adhesion to the support and formation of organized structures. Swainsonine, an inhibitor of mannosidase II, had no obvious effect. Deoxymannojirimycin, an inhibitor of mannosidase I, did not prevent cell adhesion to the support and formation of monolayers or follicles, but it favored the maintenance of follicles at a time when they were no longer present in controls. It also led to the appearance of some follicles in cultures without thyrotropin. Castanospermine, an inhibitor of glucosidase I, did not prevent cell adhesion but slowed cell spreading, thus delaying monolayer formation. Pronase glycopeptides prepared from cell-surface glycoproteins were examined with respect to their behavior on concanavalin A-Sepharose. The glycopeptides from control cells displayed complex and high-mannose glycans. The content in complex glycans was decreased in inhibitor-treated cells, while that in hybrid or high-mannose glycans was increased, indicating that the inhibitors modify the N-glycan structures. In conclusion, N-glycosylation of glycoproteins is necessary for cellular adhesion to the support. Complex structures do not seem necessary for cell adhesion monolayer or follicle formation. High-mannose structures favor follicular organization, while glucoses on the high mannose structures hinder cell spreading.

Effects of deoxymannojirimycin and castanospermine on the polarized secretion of thyroglobulin.

In order to better explore a possible role of oligosaccharide structures in the polarized secretion of thyroglobulin (Tg), we labeled cultured porcine thyroid cells with L-[3,4,5-3H]leucine or D-[2-3H]mannose in the presence or absence of the following inhibitors of N-linked oligosaccharide processing: deoxymannojirimycin (dMM), an inhibitor of mannosidase I expected to give rise to high mannose units exclusively, thus abolishing the formation of lactosaminyl branches, and castanospermine (Cs), an inhibitor of glucosidases which is expected to produce glucosylated high mannose oligosaccharides. [3H]Leucine pulse-chase experiments were performed to study the rate of Tg secretion in the presence and absence of dMM (1 mM) or Cs (1.6 mM). After a 15-h chase period, dMM and Cs did not modify the relative proportions of released Tg (65% in the follicular content, 16% in the medium) and cellular Tg (19%). In contrast, Cs led to a lower rate of secretion in both secretory pathways (t1/2 increased from 42 min to 105 min in the apical pathway and from 108 min to 138 min in the basal pathway). After a long-term labeling (16 h) with [3H]leucine or [3H]mannose, drugs did not notably affect the relative proportions of labeled Tg in the different compartments (follicular content, medium, and cell). The structures of N-glycans borne by Tg, with and without drugs, were checked. After Tg immunoprecipitation, pronase-glycopeptides were fractionated on concanavalin A-Sepharose 4B. The glycopeptides tightly bound to the lectin were treated by endo-beta-N-acetylglucosaminidase H and oligosaccharides separated by HPLC. With dMM, complex-type glycans of Tg were totally replaced by high mannose-type glycans, Man8-9GlcNAc; Cs induced the accumulation of glucosylated high mannose-type structures, Glc3Man7-9GlcNAc and Glc2Man8-9GlcNAc, but the action of this inhibitor was not total. In conclusion the correct secretion of Tg does not require the presence of the sialyllactosaminyl structure; the presence of glucose residues on high mannose-type structures lowers the rate of exocytosis.

Binding and internalization of thyroglobulin: selectivity, pH dependence, and lack of tissue specificity.

Thyroglobulin (Tg), the precursor of thyroid hormones, follows a unique secretion, storage, and recapture pathway. The first steps of recapture were studied by investigating the binding of 125I-labeled Tg on the apical surface of inside-out follicles and its internalization. The selectivity of the process was assessed by using molecules other than Tg and/or nonthyroid cells. Tg binding to the apical surface of thyroid inside-out follicles is selective relative to the binding of other molecules. It increases sharply over pH 8.0 and occurs through specific sites of moderately high affinity (Kd = approximately 200 nM; 2 x 10(4) sites/cells). At pH < 8.0 it occurs through numerous sites of very low affinity considered nonspecific. Endocytosis, although weak under our conditions, increases at pH 8.0 concomitantly with binding. Over pH 8.2, however, some inhibition occurs. Surprisingly, Tg binding and endocytosis are not tissue specific, as they showed the same properties on thyroid inside-out follicles and Madin-Darby canine kidney or Chinese hamster ovary cells. Thus, a selective uptake of Tg mediates its recapture by thyroid cells. This selectivity is an intrinsic Tg property, not dependent on the thyrocyte apical surface, as it was observed with Madin-Darby canine kidney and Chinese hamster ovary cells. Given the pH effect observed, we suggest that Tg binding is a regulated phenomenon and that, through luminal pH control, it can vary from a basal level at neutral pH to a stimulated level over pH 8.0.

Calnexin and calreticulin binding to human thyroperoxidase is required for its first folding step(s) but is not sufficient to promote efficient cell surface expression.

Human thyroperoxidase (hTPO) is a type I transmembrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. In a previous study we stably expressed hTPO in Chinese hamster ovary cells and observed that after the synthesis, only 20% of the hTPO molecules were recognized by a monoclonal antibody (mAb 15) directed against a conformational structure, and that only 2% were able to reach the cell surface. In the present study it was proposed to determine how calnexin (CNX) and calreticulin (CRT) contribute to the folding of hTPO. Sequential immunoprecipitation was performed using anti-CNX or anti-CRT followed by anti-hTPO antibodies, and the results showed that CNX and CRT were associated with hTPO. Inhibiting the interactions between CNX or CRT and hTPO using castanospermine greatly reduced the first step(s) in the hTPO folding process. Under these conditions, the half-life of this enzyme was greatly reduced (2.5 vs. 17 h in the control experiments), and hTPO was degraded via the proteasome pathway. This reduced the rate of hTPO transport to the cell surface. Overexpression of CNX or CRT into the hTPO-CHO cells was found to enhance the first hTPO folding step(s) by 20-60%, but did not increase the level of hTPO present at the cell surface. All in all, these findings provide evidence that CNX and CRT are crucial to the first step(s) in hTPO folding, but that interactions with other molecular chaperones are required for the last folding steps to take place.

Human thyroperoxidase is largely retained and rapidly degraded in the endoplasmic reticulum. Its N-glycans are required for folding and intracellular trafficking.

Human thyroperoxidase (hTPO), a type I transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis and plays a major role in thyroid autoimmunity. Whereas hormonosynthesis occurs at the apical membrane of thyroid cells, TPO localizes mainly in the perinuclear membrane and the endoplasmic reticulum. To establish the intracellular trafficking and the structural characteristics of hTPO in the various cell compartments, hTPO was stably expressed in the Chinese hamster ovary cell line, and its folding was studied with two monoclonal antibodies (mAbs): mAb 47, recognizing a linear epitope; and mAb 15, recognizing a conformational epitope present in the mature protein. The results show that only 15-20% of hTPO molecules were able to acquire a conformation suitable for the recognition by mAb 15. On the other hand, only a part (approximately 15%) of the latter were able to reach the plasma membrane. The hTPO, unable to fold correctly, was more rapidly degraded than that recognized by mAb 15 (half-time, 2 h vs. 7 h). Study of the carbohydrate content of hTPO showed that N-glycans with complex-type structure were found only on hTPO at the cell surface, whereas intracellular hTPO bore high-mannose-type structures. Taken together, these data demonstrate that the intracellular pool of enzyme is formed of newly synthesized molecules and is not caused by recycling of mature hTPO from the cell surface. Complete inhibition of hTPO N-glycosylation with tunicamycin led to a 95% decrease in hTPO at the plasma membrane and, thus, to a decrease in enzymatic activity at the cell surface, emphasizing the role of N-glycans in the intracellular trafficking of hTPO. However, inhibition of formation of complex-type structures with deoxymannojirimycin and of O-glycans with phenyl-alpha-GalNAc did not influence the intracellular trafficking and enzymatic activity of hTPO.

Glycosylation of thyroglobulin secreted by porcine cells cultured in chamber system: thyrotropin controls the number of oligosaccharides and their anionic residues.

Porcine thyroid cells were cultured in porous bottom chambers in the presence or in the absence of TSH added to the basal medium. Radiolabeled-sugar (3H-mannose) was added to the basal medium on day 11 for 4 days and the glycosylation of thyroglobulin (Tg), the major glycoprotein secreted into the apical medium, was studied. The incorporation of 3H-mannose per molecule of Tg was increased 1.5-fold by a 50 microU/ml minimal concentration of TSH. The distribution of glycopeptides (after pronase digestion) on concanavalin A sepharose column was not modified by the presence of TSH. However this distribution was different from that observed for Tg extracted from gland (more multiantennary units than biantennary units and polymannose units). After desialylation and desulfation, the sizes of the oligosaccharide chains analyzed on HPLC appeared similar when cells were cultured under stimulation or not. Thus TSH enhanced sugar incorporation without modifying either the distribution of the different oligosaccharide moieties or their sizes. Consequently the effect of TSH was a 1.5-fold increase in oligosaccharide chains linked to asparagine residues. 3H-Mannose-oligosaccharide chains were then analyzed on ion-exchange HPLC before and after desialylation and desulfation. The number of anionic residues per oligosaccharide unit (particularly sulfate residues) was higher in the absence of TSH than in the presence of TSH. Nevertheless, since TSH increased the number of carbohydrate units per molecule of Tg 1.5-fold, the total content of anionic residues bound to oligosaccharide units per molecule of Tg seems not to be modified by TSH.

The number of oligosaccharides borne by porcine thyroglobulin is variable.

Purified porcine thyroglobulin (Tg) was fractionated on a concanavalin A-Sepharose 4B column by a step-wise elution with increasing concentrations of methyl alpha-mannoside (fraction A, 50 mM; B, 100 mM; C, 200 mM; D, 500 mM, and E, 1 M), and its fractional ratio was 12.8:28.6:26.4:19.7:12.4. These five fractions showed the same profile in polyacrylamide gel electrophoresis. The subfractions were analyzed for their relative contents in oligosaccharides of each structure type and for their monosaccharide contents. In fractions B, C, D, and E the former varied between 15-22% for triantennary complex-type, 47-60% for biantennary complex-type, and 22-30% for high mannose-type oligosaccharide. Fraction A showed a higher percentage of triantennary complex-type structures (36%) and a lower percentage of biantennary complex-type structures (17%). The monosaccharide numbers increased from fraction A to E: 85 to 135 mannose residues, 60 to 82 galactose residues, 84 to 115 N-acetyl glucosamine residues, and 22 to 28 sialic acid residues. After analysis of the number of mannose residues contained in the high mannose-type structures, it was possible to calculate the number of oligosaccharides borne by each Tg subfraction. This number was approximately the same for fractions A and B (22.4 and 21.7), then it increased from B to E (21.8 to 32.9). These results account for the separation obtained on the concanavalin A-Sepharose 4B column. Separation of the two first subfractions bearing the same number of oligosaccharides is certainly due to the higher number of high mannose-type structures in B. In conclusion, the studies reported here show that porcine Tg is heterogeneous, and mainly so in terms of total number of N-glycan structures.