SMED-TLX-1 (NR2E1) is critical for tissue and body plan maintenance in Schmidtea mediterranea in fasting/feeding cycles.

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

Valproic acid, a molecular lead to multiple regulatory pathways.

Valproic acid (2-propyl pentanoic acid) is a drug used for the treatment of epilepsy and bipolar disorder. Although very rare, side effects such as spina bifida and other defects of neural tube closure indicate that valproic acid interferes with developmental regulatory pathways. Recently obtained data show that valproic acid affects cell growth, differentiation, apoptosis and immunogenicity of cultured cancer cells and tumours. Focused studies uncovered the potential of valproic acid to interfere with multiple regulatory mechanisms including histone deacetylases, GSK3 alpha and beta, Akt, the ERK pathway, the phosphoinositol pathway, the tricarboxylic acid cycle, GABA, and the OXPHOS system. Valproic acid is emerging as a potential anticancer drug and may also serve as a molecular lead that can help design drugs with more specific and more potent effects on the one side and drugs with wide additive but weaker effects on the other. Valproic acid is thus a powerful molecular tool for better understanding and therapeutic targeting of pathways that regulate the behaviour of cancer cells.

Supplementary nuclear receptor NHR-60 is required for normal embryonic and early larval development of Caenorhabditis elegans.

The C. elegans genome encodes an unexpectedly large number of NHRs, the majority of which are classified as supplementary nuclear receptors (supnrs) that are likely to have evolved from an ancestral protein related to vertebrate HNF-4. To understand the need for this large repertoire of potential ligand-activated transcription factors, we have begun to study an 18-member subgroup defined by DNA binding domain relatedness. Here we report on NHR-60, a supnr expressed ubiquitously throughout development with a distinct pattern of localization on the nuclear periphery. Both antibody staining and GFP reporter genes demonstrated high-level expression and accumulation of NHR-60 in seam cell nuclei that is dependent on NHR-23 activity. Interference with NHR-60 activity, by either RNAi or overexpression of a putative dominant negative isoform, results in embryonic and early larval lethality, including defects in seam cell development. This adds NHR-60 to the list of C. elegans NHRs playing important roles in development.

BIR-1, the homologue of human Survivin, regulates expression of developmentally active collagen genes in C. elegans.

BIR-1 and Survivin are highly conserved members of the inhibitor of apoptosis protein family that regulate cell division in nematodes and mammals and inhibit apoptosis in mammals. In the C. elegans genome, bir-1 is organized in an operon together with transcription and splicing cofactor CeSKIP (skp-1) and is highly expressed during embryogenesis as well as in non-dividing cells during larval development. Previously we have shown that BIR-1 regulates transcription and development and its loss-of-function phenotype overlaps with loss of function of CeSKIP and nuclear hormone receptor CHR3 (NHR-23). Here we searched for genes whose expression is affected by BIR-1 loss of function using whole-genome microarray experiments and identified several collagen genes as candidate targets of bir-1 inhibition in L1 larval stage. The decreased expression of selected collagen genes in bir-l-inhibited larvae was confirmed by quantitative RT-PCR. Next, we generated transgenic lines expressing bir-1 mRNA under a heat shock-regulated promoter and tested whether bir-1 overexpression has the potential to augment the expression of genes that showed decreased expression in worms treated with bir-1 RNAi. Overexpression of bir-1 resulted in a pronounced increase (2 to 5 times) of the expression of these genes. Our findings support the concept that BIR-1, a protein generally regarded as a mitotic factor, is involved in the regulation of transcription during normal development of C. elegans and has a strong ability to affect transcription of developmentally active genes if overexpressed.

Elevated and deregulated expression of HDAC3 in human astrocytic glial tumours.

Abnormal expression of histone deacetylases may contribute to the establishment of a cancer specific transcription profile. We examined expression of HDAC3 in human non-malignant gliosis and glial astrocytic tumours. Samples from four non-malignant gliosis and 17 astrocytic gliomas (six of grade II, one of grade III and ten of grade IV) removed for therapeutic purposes were assayed for HDAC3 expression at mRNA and protein levels. HDAC3 mRNA was detected in non-tumorous gliosis as well as in all examined glial tumours. Seven out of eleven examined high-grade tumours showed an elevated number of copies of HDAC3 mRNA. Western blot analysis detected high levels of expression of HDAC3 in the majority of the examined tumours. Immunohistochemistry and immunofluorescence made on a collection of 35 astrocytic tumours detected nuclear as well as cytoplasmic HDAC3 expression in all of those tumours. While the distribution of HDAC3 was both nuclear as well as cytoplasmic and moderate in intensity in non-malignant tissues and low-grade gliomas, high-grade tumours expressed HDAC3 in a focally deregulated pattern that included strongly pronounced cytoplasmic localization. Confocal microscopy and additional co-localization analysis detected nuclear HDAC3 in all tumours examined. We conclude that HDAC3 expression is elevated in human astrocytic tumours and its expression pattern is deregulated at the cellular level in high-grade gliomas.

Expression of CD44v6 correlates with cell proliferation and cellular atypia in urothelial carcinoma cell lines 5637 and HT1197.

CD44 comprises a family of membrane adhesion molecules encoded by a single gene and diversified by alternative splicing and extensive posttranslational modifications. Alterations of CD44 expression patterns are linked to tumour invasion and formation of metastases. However, CD44 expression and its relation to the biological properties of tumours vary depending on the tumour type and origin. In transitional cell carcinoma of the urinary bladder, low CD44 expression is linked to enhanced tumour aggressiveness. We studied CD44 expression in two urothelial cancer cell lines, HT1197 and 5637. CD44s and a v6 variable exon-containing splice variants were detected in both cell lines by reverse transcription-PCR and by commercially available monoclonal antibodies. In both cell lines, Western blot analysis detected immunoreactive proteins with approximate sizes 70-85 kD, 95-110 kD, and 120-140 kD with CD44v6 antibody and weak bands with size 70-98 kD with CD44s antibody. At the cellular level, the pattern of CD44 immunoreactivity correlated with a lower level of cell differentiation and a higher degree of cell proliferation. In HT1197 cells, the CD44v6 was detected predominantly in small proliferating cells and in large multinuclear atypical cells. CD44s and CD44v6 displayed low immunoreactivity in HT1197 cells with a higher degree of epithelial differentiation. The 5637 cells expressed CD44v6 strongly and CD44s weakly. We conclude that CD44v6 expression correlates with a higher proliferative activity and with a stem cell-like phenotype in both cell lines and with cellular atypia in HT1197 cells.

Analysis of the thyroglobulin internalization process using in vitro reconstituted thyroid follicles: evidence for a coated vesicle-dependent endocytic pathway.

We have designed a new experimental system based on in vitro reconstituted thyroid follicles (RTF) to study the relative implication of macropinocytosis and micropinocytosis processes in the internalization of thyroglobulin (Tg). Thyrocytes cultured in the presence of TSH reorganize in histiotypic and functional follicles. Tg, which accumulates into the newly formed intrafollicular lumen (IL), was pulse labeled with [125I]iodide. Basal or TSH-activated Tg internalization, i.e. transfer from IL to cells, was assessed by measuring [125I]Tg in the cells and the IL; the IL fraction was collected after selective opening of lumina by a short treatment of RTF in a calcium-free medium. We used the ratio between cellular and IL labeled Tg contents as an endocytic index. TSH caused a very rapid increase in the cellular uptake of labeled Tg; the endocytic index increased by a factor of 4-8. The TSH effect was maximum after 15-20 min. TSH had no effect when the chase-incubation was performed at 4 C, but exhibited the same stimulatory action in terms of both time course and amplitude of action at 20 and 37 C. The macropinocytosis-related cellular structures, the pseudopods, were never observed in RTF maintained at 20 C; they were rare at 37 C and only found after 30 min of TSH treatment. At 20 as well as 37 C, the action of TSH on Tg endocytosis was concentration dependent in the range of 0.05-10 mU/ml. A fraction of Tg internalized by thyrocytes was found in coated vesicles. The labeled Tg content of purified coated vesicles varied with the temperature of the chase-incubation and was increased in TSH-treated RTF. Taken together, these data show that endocytosis of Tg by thyroid follicular cells in resting or moderately activated states does not proceed via the pseudopod formation-dependent mechanism, also termed macropinocytosis. Tg internalization would be related to what is referred as micropinocytosis and would involve a coated vesicle-dependent endocytic pathway.

Thyroglobulin internalized by thyrocytes passes through early and late endosomes.

We have tried to characterize the intracellular compartments involved in the traffic of the thyroid prohormone thyroglobulin (Tg) from the site of storage, the follicular lumen, to the expected site(s) of proteolytic degradation, lysosomes. Electron microscope immunogold labeling with antibodies against Tg, cation-independent mannose-6-phosphate receptor (MPR), or arylsulfatase-A (ArS-A) was used to identify endocytic structures. The implication of these structures in the transport of Tg was analyzed by following the internalization and intracellular fate of Tg-colloidal gold complexes microinjected into the thyroid follicular lumen. Immunogold labeling was performed on ultrathin cryosections of intact pig tissue, in vitro reconstituted thyroid follicles (RTF), and isolated vesicles prepared by differential and isopycnic centrifugation. Microinjection experiments were carried out on RTF. Using double labeling for MPR and ArS-A, we characterized three types of structures: those slightly positive for MPR and ArS-A, those strongly positive for both markers, and those only positive for ArS-A. These compartments exhibited the properties of early endosomes (EE), late endosomes (LE), and lysosomes (L), respectively. Tg immunoreactivity was high in EE, low in LE, and undetectable in L. Similar morphological and immunochemical characteristics of EE, LE, and L were found in intact tissue, RTF, and isolated vesicles. Tg-gold complexes microinjected into the lumen of RTF were efficiently internalized within 5 min into structures with the appearance of EE. Sixty minutes after the injection, Tg-gold complexes were detected into LE and L. We present here the first direct experimental evidence for an involvement of endosomal compartments in the Tg internalization/degradation pathway. The data indicate that internalized Tg molecules are transported to EE and then transferred from EE to LE.

Endocytosis of albumin and thyroglobulin at the basolateral membrane of thyrocytes organized in follicles.

Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the circulation.

Thyroglobulin molecules internalized by thyrocytes are sorted in early endosomes and partially recycled back to the follicular lumen.

Thyroglobulin (Tg) molecules stored in thyroid follicle lumens are heterogeneous in terms of iodine and hormone contents. It has been suggested that thyroid hormone is preferentially produced from the most highly iodinated Tg molecules and that thyrocytes are capable of selecting these molecules. The cellular localization as well as the molecular basis of such a selection process are not known. The present work was undertaken to determine whether there is selectivity at the step of endocytosis and, if not, to discover other possible mechanisms. Studies were conducted on reconstituted thyroid follicles (RTF) in culture. We compared the ability of thyrocytes to internalize Tg and an exogenous protein, BSA, which is neither iodinated nor glycosylated. To identify the protein, Tg and BSA were coupled to gold particles of different size and microinjected in a fixed ratio into the lumen of RTF. Neither of the two protein gold probes detected by transmission electron microscope bound at the cell surface, and both entered the cells at a similar rate and were concentrated in early endosomes. After 20 min, both Tg-G and BSA-G were segregated into distinct vacuolar structures. At 60 min, the intracellular content of BSA-G (mainly in prelysosomes and lysosomes) was 2- to 3-fold higher than that of Tg-G. At the same time, there was a marked reduction in the BSA-G/Tg-G ratio in the lumen. The differences between the Tg-G and BSA-G distribution patterns that were amplified in TSH-treated RTF are in keeping with a back-transfer of internalized Tg toward the lumen. The existence of a cell to lumen transport of previously endocytosed Tg was further documented using intralumenal [125I]Tg as a marker. RTF pulse labeled with tracer amounts of [125I]iodide were shortly incubated with TSH to induce [125I]Tg endocytosis, and the fate of internalized [125I] Tg was studied in a chase incubation period of up to 4 h. At 20 C, where the degradation of internalized Tg is blocked, we observed a time-dependent decrease in intracellular [125I]Tg and a corresponding increase in the lumenal [125I]Tg content. This cell to lumen [125I]Tg transfer was inhibited by primaquine. In conclusion, our data show that 1) the thyroid apical endocytic process does not exhibit selectivity for Tg; 2) the thyrocyte possesses a sorting machinery for endocytosed ligands; and 3) internalized Tg molecules can be recycled back to the follicular lumen.(ABSTRACT TRUNCATED AT 400 WORDS)

A divergent role of COOH-terminal domains in Nurr1 and Nur77 transactivation.

Orphan nuclear receptors such as Nurr1 and Nur77 have conserved amino acid sequences in the zinc finger DNA binding domains and similar COOH-terminal regions, but have no known ligands. These receptors can bind DNA sequences (response elements) as monomers and can also heterodimerize with the retinoid X receptor to activate transcription. We report here the identification and initial characterization of a novel COOH-terminal truncated isoform of Nurr1, Nurr1a. Internal splicing of Nurr1 generates a frameshift such that a stop codon is prematurely encoded resulting in a naturally occurring COOH-terminal truncation. Embryonic and postnatal mouse brain showed both Nurr1 and Nurr1a mRNAs expressed during development. To characterize essential COOH-terminal elements that may be deleted from Nurr1a and determine function in putative ligand binding, we created COOH-terminal deletion mutants. Nurr1, Nur77, and 3'-truncated mutants bind in gel mobility shift assays to the monomeric Nur77 response element (B1A-RE). However, in transient transfection assays, a truncation of as little as 15 Nurr1 COOH-terminal amino acids diminished transcriptional activation of B1A-thymidine kinase-chloramphenicol acetyltransferase reporter. This result was not seen for a similar Nur77 deletion mutant, Nur77-586. Unlike full-length Nurr1 and Nur77, transactivation by Nur77-586 was not augmented in response to the presence of retinoid-like receptor and 9-cis-retinoic acid. Thus, the interaction of putative ligand binding and transactivation for Nurr1 and Nur77 may function differently.

What's new in the importance of receptors in pathology?

Highly organized multicellular organisms require a complex system of intercellular communication to coordinate the great variety of physiological functions. Major components of this system are the (i) signalling substances delivering the information and (ii) the corresponding cellular receptors recognizing the information and translating them to special intracellular organelles of the target cell. For the process of signal binding and transmission various types of receptor proteins are known playing a key role in the mutual understanding between cells. The present paper comprehends lectures on functions and deficiencies of the different types of cell receptors given during the Symposion on "Receptors in Pathology" during the 11th European Congress of Pathology. Each section is written under the personal responsibility of the author indicated.

[Preparation of colloidal gold and its macromolecular complexes]. / Príprava koloidního zlata a jeho komplexu s makromolekulami.

A critical presentation of basic methods for the preparation of colloid gold and of gold particle complexes with macromolecules to be used on the ultrastructural level.

[Use of colloidal gold in ultrastructural cytochemistry]. / Vyuzití koloidního zlata v ultrastrukturální cytochemii.

Colloid gold stabilized by macromolecules with various binding characteristics proved to be usable in transmission electron microscopy: Nucleic acids localization was studied in tissue cultures after embedding by techniques autoimmune serum-protein A--gold and RNase--gold. Technique with serum autoantibodies against double-stranded DNA resulted in marking condensed chromatin. Further experiments with ultrastructural localization of other autoimmune components seemed to be perspective and of diagnostic importance. Technique with RNase--gold complex marked especially ribosomes, nucleolus and interchromatin nuclear spaces. Herpes virus, rotavirus and enterovirus were identified in negative contrast by the technique antivirus serum--protein A--gold. A higher warrant of evidence achieved by the method may be used in virologic diagnosis. A small amount of tubulin was found in isolated permeabilized nuclei of Xenopus laevis by a direct immunocytochemical method with complex monoclonal IgG against tubulin-gold. Binding of complex of triiodothyronine-bovine serum albumin--gold on the cytoplasmic membrane of LEP cells in a short term tissue culture showed a possibility of tracing non-peptidic hormones binding on specific receptors.