Metabolism of dehydroepiandrosterone sulfate and estrone-sulfate by human platelets.

The aim of the present research was to study the uptake of DHEAS, and to establish the intracrine capacity of human platelets to produce sex steroid hormones. The DHEAS transport was evaluated through the uptake of [(3)H]-DHEAS in the presence or absence of different substrates through the organic anion transporting polypeptide (OATP) family. The activity of sulfatase enzyme was evaluated, and the metabolism of DHEAS was measured by the conversion of [(3)H]-DHEAS to [(3)H]-androstenedione, [(3)H]-testosterone, [(3)H]-estrone and [(3)H]-17beta-estradiol. Results indicated the existence in the plasma membrane of an OATP with high affinity for DHEAS and estrone sulphate (E(1)S). The platelets showed the capacity to convert DHEAS to active DHEA by the steroid-sulfatase activity. The cells resulted to be a potential site for androgens production, since they have the capacity to produce androstenedione and testosterone; in addition, they reduced [(3)H]-estrone to [(3)H]-17beta-estradiol. This is the first demonstration that human platelets are able to import DHEAS and E(1)S using the OATP family and to convert DHEAS to active DHEA, and to transform E(1)S to 17beta-estradiol.

Development of high-throughput tools to unravel the complexity of gene expression patterns in the mammalian brain.

Genomes of animals contain between 15000 (e.g. Drosophila) and 50000 (human, mouse) genes, many of which encode proteins involved in regulatory processes. The availability of sequence data for many of these genes opens up opportunities to study complex genetic and protein interactions that underlie biological regulation. Many examples demonstrate that an understanding of regulatory networks consisting of multiple components is significantly advanced by a detailed knowledge of the spatiotemporal expression pattern of each of the components. Gene expression patterns can readily be determined by RNA in situ hybridization. The unique challenge emerging from the knowledge of the sequence of entire genomes is that assignment of biological functions to genes needs to be carried out on an appropriately large scale. In terms of gene expression analysis by RNA in situ hybridization, efficient technologies need to be developed that permit determination and representation of expression patterns of thousands of genes within an acceptable time-scale. We set out to determine the spatial expression pattern of several thousand genes encoding putative regulatory proteins. To achieve this goal we have developed high-throughput technologies that allow the determination and visualization of gene expression patterns by RNA in situ hybridization on tissue sections at cellular resolution. In particular, we have invented instrumentation for robotic in situ hybridization capable of carrying out in a fully automated fashion, all steps required for detecting sites of gene expression in tissue sections. In addition, we have put together hardware and software for automated microscopic scanning of gene expression data that are produced by RNA in situ hybridization. The potential and limitations of these techniques and our efforts to build a Web-based database of gene expression patterns are discussed.

Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud.

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. We have explored signal transduction pathways in the chicken limb bud. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induced RACK1 and such induction of RACK1 expression was accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region resulted in buds that did not express sonic hedgehog (Shh) and developed into wings that were severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcome the effects of blocking PKC with chelerythrine and resulted in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs.

Immunohistochemical detection of estrogen receptor alpha in articular chondrocytes from cows, pigs and humans: in situ and in vitro results.

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors alpha and beta. The goal of the present study was to search for estrogen receptor alpha in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor alpha in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor alpha is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O2 or with 5% O2 positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.

Immunoelectron microscopy in embryos.

Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.

A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor alpha in target tissues.

An intrauterine pulse-stimulation with estradiol induced changes in the subcellular localization of estrogen receptor alpha in porcine endometrium, as detected with F(ab') fragments of various anti-receptor antibodies covalently linked to nanogold. The low-sterically hindered immunoreagents--recognizing different epitopes within the hormone binding domain--allowed for an efficient immunolabeling of estradiol receptor alpha, detecting it both in the cytoplasm and the nucleus of nonstimulated epithelium cells. In the cytoplasm, the receptor often seemed to be associated with actin filaments and the endoplasmatic reticulum. After the stimulation with estradiol, a predominantly nuclear localization and a labeling of nucleoli was observed. Our immunoelectron microscopy study demonstrates a localization of the receptor in cytoplasmic organelles that increased after the hormone pulse. These organelles exhibited the morphological properties of lysosomes and relocated to the perinuclear area. In analogous cytoplasmic organelles, the presence of cathepsin D was detected via indirect immunogold labeling, justifying their classification as lysosomes. Quantitative examinations revealed that not only the number of lysosomes in the proximity of the nucleus but also their immunostaining for estradiol receptor alpha increased significantly after the hormone pulse. Thus, estradiol induces both the rapid shift of receptor into the nucleus, a slower perinuclear accumulation of lysosomes and an increase of lysosomal ERalpha-immunoreactivity. These results suggest a role for lysosomes in the degradation of receptor shuttling out of the nucleus. This could serve as termination of the estradiol receptor alpha-dependent activation of target cells. This hypothesis is strengthened by the fact that the receptor content in uterine tissue declined drastically few hours after the hormone pulse.

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter.

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

Identification of target cells by immunohistochemical detection of covalently rearranged estradiol in rehydrated paraffin sections.

Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.

Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation.

Mouse-virulent Salmonella typhimurium strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis, csgBA(C) and csgDEFG, from S. typhimurium SR-11 revealed the same gene order and flanking genes as in Escherichia coli. The divergence of the curli region between S. typhimurium and E. coli at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently, S. typhimurium genes on low-copy-number plasmids were able to complement respective E. coli mutants, although not always to wild-type levels. rpoS and ompR are required for transcriptional activation of (at least) the csgD promoter. The high degree of conservation at the protein level and the identical regulation patterns in E. coli and S. typhimurium suggest similar roles of curli fibers in the same ecological niche in the two species.

Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

Binding and distribution of Na2B12H11SH on cellular and subcellular level in tumor tissue of glioma patients in boron neutron capture therapy.

To determine binding and distribution of Na2B12H11SH (BSH) in glioma tissue in case of boron neutron capture therapy, an antibody to this compound was produced and used in immunohistochemical investigations. It is possible to trace BSH in immunohistochemistry, because BSH is firmly bound to the glioma tissue. The antibody against BSH is specific for that antigen, as tumor tissue from patients without BSH administration did not stain. In areas of healthy brain from BSH infused patients, no staining of tissue was detectable. In tumor tissues, BSH is presenting as a strong staining in cytoplasm and nucleus areas.

The 23-kDa light-stress-regulated heat-shock protein of chenopodium rubrum L. is located in the mitochondria.

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et al. (1995, Biochem J 311:805-813) and LaFayette et al. (1996, Plant Mol Biol 30:159-169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions.

TSH-receptor expression and human thyroid disease: relation to clinical, endocrine, and molecular thyroid parameters.

Thyrotropin receptor (TSH-R) gene expression can be positively or negatively regulated by TSH and stimulating TSH-R antibodies (TSAbs) in immortalized thyroid cell lines such as rat FRTL-5 cells. However, regulation is less clear in other mammalian cells including cultures of human thyroid cells. Additionally, it has been suggested, based on FRTL-5 cell data, that TSH-R gene negative regulation by TSH or TSAbs might be lost in Graves' disease. The present study evaluated TSH-R gene transcript levels in thyroids from patients with Graves' disease to correlate in vivo data with in vitro observations or hypotheses. TSH-R mRNA levels were characterized in a total of 66 human thyroid glands with particular concern to levels in Graves' patients. Results were related to clinical parameters, transcript levels of thyroglobulin (TG), and thyroid peroxidase (TPO), as well as transcript levels of thyroid transcription factor 1 (TTF-1) which regulates the expression of all three genes and paired box-gene 8 (Pax-8) which regulates TG and TPO gene expression. Northern blot analyses showed that TSH-R expression was significantly increased, 2.2-fold, in Graves' thyroids (p = 0.0098, n = 35) by comparison to normals (n = 6). TSH-R mRNA levels were decreased to 30% and 7% of normal levels in Hashimoto's thyroids (p = 0.0281, n = 5) and anaplastic carcinomas (p = 0.0033, n = 6), respectively. No significant changes were seen in endemic goiters (n = 8) and in thyroid autonomy (n = 6). TSH-R RNA levels were higher, 3.6-fold, in thyroids of a subgroup of Graves' patients that had not been pretreated with iodide before surgery (n = 10) by comparison to thyroids from those that had been treated before surgery, 1.7-fold (n = 25). TSH-R antibodies exhibited a nonsignificant tendency toward a negative correlation. All other clinical or endocrine parameters showed no clear relation to TSH-R mRNA levels. Pax-8 and TTF-1 transcripts were detectable in normal thyroids; however, Pax-8 expression was increased in Graves' thyroids (3.8-fold), whereas TTF-1 expression was only minimally changed in all thyroids investigated. Changes of the two did not correlate. Pax-8 expression correlated with TG and TPO expression (in all cases, p = 0.0001); TTF-1, despite its minimal change, still correlated with TG (p = 0.0471) but not with TPO expression (p = 0.0984). TTF-1, again despite its minimal changes, correlated positively with TSH-R gene expression (p = 0.0251); however, surprisingly, Pax-8, which does not regulate TSH-R gene expression, correlated even better with TSH-R transcript levels (p = 0.0001). We conclude that augmentation of TSH-R expression levels, and thus potential ligand binding sites, may indicate an important regulatory principle in the pathogenesis of autoimmune hyperthyroidism in vivo: the responsiveness of the TSH-R to TSH and TSAb induced negative regulation is lost. This increase of TSH-R expression levels is not due to an ongoing transcriptional activation of the TTF-1 gene. Pax-8, though positively correlated with TSH-R RNA levels, cannot be the factor either, because Pax-8 does not upregulate TSH-R expression. This predicts that other factors involved in TSH-R induced negative regulation are abnormal and must be searched for and evaluated.

Electron spectroscopic imaging of antigens by reaction with boronated antibodies.

Two small homogeneous markers for electron spectroscopic imaging (ESI) containing eight dodecaborane cages linked to a poly-alpha, epsilon-L-lysine dendrimer were synthesized; one of these was made water soluble by the attachment of a polyether. The markers were coupled to the sulfhydryl group of (monovalent) antibody fragments (Fab') by a homobifunctional cross-linker. While the coupling ratios of the poorly water-soluble compound did not exceed 20%, the polyether-containing variant reacted quantitatively. Its suitability for immunolabelling was tested in a study of the mechanism of the transcellular transport of an administered heterologous protein (bovine serum albumin, BSA) through ileal enterocytes of newborn piglets by endocytotic vesicles in comparison to conventional immunogold reagents. The post-embedding technique was employed. The boronated Fab' gave rise to considerably higher tagging frequencies than seen with immunogold, as could be expected from its form- and size-related physical advantages and the dense packing of BSA in the vesicles. The new probe, carrying the antigen-combining cleft at one end and the boron clusters at the opposite end of the oval-shaped conjugate, add to the potential of ESI-based immunocytochemistry.

Immunocytochemistry by electron spectroscopic imaging using a homogeneously boronated peptide.

A linear all-L-oligopeptide containing five carboranyl amino acids (corresponding to 50 boron atoms) was synthesized and specifically attached to the free thiol group of monovalent antibody fragments F(ab)'. The boronated immunoreagent was used for the direct post-embedding detection of somatotrophic hormone in ultrathin sections of porcine pituitary embedded in Spurr resin. The specific boron-labelling of secretory vesicles in somatotrophs was detected by electron spectroscopic imaging and confirmed by conventional immunogold labelling run in parallel. In comparison with immunogold, boron-labelled F(ab)'-fragments showed higher tagging frequencies, as was expected; the small uncharged immunoreagents have an elongated shape and carry the antigen-combining structure and the detection tag at opposite ends, thus allowing for high spatial resolution in electron spectroscopic imaging.

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

Immunocytochemistry by electron spectroscopic imaging using well defined boronated monovalent antibody fragments.

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI)--an electron microscopic technique with high-resolution potential. Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not affected by the attachment of the markers and the resulting immunoprobes exhibited an elongated shape with the antigen combining site and the label located at opposite ends. The labeling densities observed with these reagents were found to be significantly higher than those obtained by using conventional colloidal gold methods. Combined with digital image processing and analysis systems, boron-based ESI proved to be a powerful approach in ultrastructural immunocytochemistry employing pre- and post-embedding methods.

Identification by affinity chromatography of boar sperm membrane-associated proteins bound to immobilized porcine zona pellucida. Mapping of the phosphorylethanolamine-binding region of spermadhesin AWN.

We have identified boar sperm membrane components recovered by affinity chromatography on a porcine zona pellucida affinity column. The major zona pellucida-bound proteins were spermadhesins AWN and AQN-3, the heparin-binding protein pAIF, and a homolog of the mouse milk fat globule membrane protein. All these proteins are phospholipid-binding proteins peripherally associated with the plasma membrane. Our data suggest that coating proteins tightly bound to the external lipid bilayer may act as major zona pellucida-binding molecules. Using a synthetic peptide approach we show that the regions of spermadhesin AWN comprising residues 6-12 and 104-108 possess affinity for phosphorylethanolamine. These two amino acid sequences are in close proximity in the predicted structural model for AWN, and in opposite location to its carbohydrate-recognition domain. Taken together, our data provide further evidence for the possible involvement of members of the porcine spermadhesin protein family in gamete interaction and suggest a model for the ultrastructural disposition of functional domains of spermadhesin AWN bound to the sperm surface.

Functional and morphological changes of the thyroid gland following 5 days of pulsatile TRH stimulation in male rats.

The aim of the present study was to evaluate in vivo the selective effects of a small increase in plasma TSH levels on thyroid function, proliferation and morphology. Chronically catheterized male Sprague-Dawley rats were stimulated i.v. over 5 days either with TRH (2 micrograms TRH in 100 microliters 0.9% (w/v) NaCl (TRH-P) or the NaCl carrier alone (P), both given as pulses every 2 h. Control groups were cotreated i.v. with 10 micrograms thyroxine (T4)/100 g body weight per day (TRH-P + T4) starting 2 days before pulsatile stimulation. TSH plasma levels were approximately doubled by TRH-P (P < or = 0.001), T4 plasma levels significantly increased (P < or = 0.001) but tri-iodothyronine plasma levels did not change compared with treatment with P. No significant changes between groups were found in thyroid weight and in intrathyroidal iodine content, but the percentage of 5-bromo-2'-desoxyuridine-labelled thyrocytes as a marker of proliferation in TRH-P-treated animals was significantly increased over P or TRH-P + T4 (P < or = 0.001). Ultrastructural analysis of the thyroid evaluated by electron microscopy revealed a significant increase in the number of lysosomes (P < or = 0.001). The size of the endoplasmic reticulum (ER) in relation to the cytoplasm was significantly increased when treated with TRH-P compared with P or TRH-P + T4 (P < or = 0.001). Post-embedding immunogold staining revealed Tg as a major product within ER cisternae. Immunogold labelling was moderate in controls and higher densities of gold particles were obtained in TRH-P-treated animals (P < or = 0.001). In conclusion, short-term pulsatile TRH stimulation increasing the plasma levels of immunoreactive TSH only twofold is capable of inducing hypertrophy of the thyrocytes by gross ultrastructural changes which are paralleled by an increase in circulating T4. These data underscore the dominant role of TSH on thyroid ultrastructure within the narrow boundaries of normal physiological regulation.

Immunogold labelling of estradiol receptor in MCF7 cells.

The distribution of estradiol receptor in serial sections of estradiol-deprived and estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of estradiol-deprived cells, the receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antireceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen receptor in estradiol-deprived cells and support the premise of an estradiol-induced translocation of this ligand-dependent transcription regulator.