Changes in colloid droplets and dense bodies in rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

Two changes in cytoplasmic granules, corresponding to colloid droplets and dense bodies, in rat thyroid follicular cells during 24 hr were analyzed morphometrically in the apical, intermediate, and basal parts of the cells. The volume and surface densities of the cytoplasmic granules and their volumes and surface areas in the apical and intermediate parts varied bimodally over a 24-hr day, being high at 1200 and 0000 hr and low at other times. In apical and intermediate regions, colloid droplets were predominant at 1200 and 0000 hr, whereas the cytoplasmic granules became heterogeneously or homogeneously electron-dense at other times. In the basal part, these morphometric parameters were not synchronized with those in the other two regions, showing lower values. The basal cytoplasmic granules were heterogeneously or homogeneously electron-dense. Small, homogeneously dense granules appeared in both the apical and intermediate parts and in the basal part with a certain time lag. These granules were often in contact with colloid droplets in the lumenal two parts at 1200 and 0000 hr. Their numerical densities were low in the apical part, but high in the intermediate and basal parts. These results suggest that newly formed colloid droplets migrate from the apical through the intermediate to the basal part, changing in appearance as they go. Moreover, it seems likely that small, homogeneously dense granules are a final form of cytoplasmic granule. They may be reused for degradation of colloidal protein.     

Immunocytochemical localization of cathepsins B, H, L, and T4 in follicular cells of rat thyroid gland

To localize cathepsins B, H, and L in follicular cells of rat thyroid gland, we applied immunocytochemistry to the thyroid tissue using their respective monospecific antibodies. On serial semi-thin sections, cathepsins B, H, and L were localized in granules of various sizes located throughout the cytoplasm, whereas T4 was detected in larger granules located in the apical and supranuclear regions. By electron microscopy, cathepsins B, H, and L were localized in large less-dense granules (so-called colloid droplets) and in dense bodies of various sizes, whereas T4 was localized more intensely in large less-dense granules than in smaller dense bodies. By double immunostaining using an immunogold method, cathepsins H and B or L were co-localized in the same cytoplasmic granules. Moreover, immunoblotting demonstrated that proteins similar to cathepsins B, H, and L in the liver are present in the thyroid gland. These results suggest that cathepsins B, H, and L participate not only in degradation of thyroglobulin but in maturation of thyroid hormones, although it remains unknown whether all of them participate in the maturation process.     

Bimodal variations in subcellular structures of rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

To clarify 24-hr variations in rat thyroid follicular cells under physiological conditions, their subcellular structures were examined at six evenly spaced times during 24 hr by using a morphometric technique. The volume, surface, and numerical densities of subcellular structures varied distinctly over each 24-hr period, with a bimodal pattern. The cellular and nuclear volumes varied also bimodally over 24 hr. A decrease in the surface density of the apical plasmalemma at 1200 and 0000 hr coincided with an increase in volume density of cytoplasmic granules representing colloid droplets and dense bodies. Most granules (colloid droplets) appearing at these times were reduced in electron density. At other times, especially at 1600 and 0400 hr, morphometric parameters of rough endoplasmic reticulum (rER), Golgi complex, and subapical vesicles were prominently increased, although values for rER did not peak at 1600 hr. At these times, the volume densities of cytoplasmic granules, most of which were heterogeneous and of homogeneous electron density, were decreased. These findings coincided with immediate and subsequent reactions of follicular cells after injection of thyroid-stimulating hormone (TSH). From the evidence, it seems likely that variations in follicular cells over a 24-hr period reflect variations in blood TSH concentration. The total membrane areas of membrane components in follicular cells were calculated from the morphometric measurements. These areas fluctuated unimodally during 24 hr over a 65% range. This suggests that the membranes in follicular cells are subjected to cyclic degradation and regeneration during each 24-hr period.     

LOCALIZATION OF ESTERASE AND ACID PHOSPHATASE IN GRANULES AND COLLOID DROPLETS IN RAT THYROID EPITHELIUM

Droplets which stain like colloid occur in the cytoplasm of the thyroid follicular epithelium of the rat following stimulation of the gland by thyroid-stimulating hormone (TSH). The occurrence of droplets was remarkably reduced when the lumen became depleted of colloid. Acid phosphatase and esterase were localized in the thyroid droplets and, in addition, in granules largely around the nucleus. Stimulation by TSH resulted in an increase in the number of droplets containing enzyme. Twenty-four hours after hypophysectomy, enzyme-associated granules were localized at the basal end of the cell and droplets were absent. Intravenous injection of TSH resulted in formation of droplets at the apical end of the cell and migration of enzyme-associated granules toward the apical end of the cell. The droplets were first observed approximately 10 minutes after TSH administration and at this time did not appear to contain enzyme. Within 15 minutes many droplets contained enzyme. The granules were largely localized near the nucleus on its apical side 30 minutes after a dose of 25 milliunits of TSH, but were less well localized following one-tenth this dose. These results indicate that the epithelial cell of the thyroid gland contains preformed hydrolytic enzymes associated with granules (lysosomes). When the gland is stimulated by TSH, droplets are formed from colloid derived from the lumen (phagosomes), and hydrolytic enzymes are transferred from granules to the droplets. The droplets may be intracellular organelles for hydrolysis of colloid and liberation of thyroxine prior to the release of thyroxine into the blood.     

Correlation of 24-hour fluctuations in renin granules of juxtaglomerular cells and in renin and angiotensinogen in blood plasma of the rat

Summary Subcellular structures of juxtaglomerular (JG) cells in the rat kidney were morphometrically examined at six evenly spaced times over 24 h. Plasma renin activities and angiotensinogen concentrations were also measured at these times. The cell volumes were larger at 20.00 h and 04.00 h than at 00.00 h, whereas the nuclear volumes peaked at 20.00 h and 08.00 h, decreasing at 00.00 h and 16.00 h. The volume and surface densities of renin granules and their individual volumes and surface areas peaked at 16.00 h and 00.00 h, decreasing at 20.00 h and 08.00 h, whereas their numerical densities peaked at 20.00 h, decreasing at 12.00 h. The surface densities of the rough endoplasmic reticulum (rER) peaked at 20.00 h, decreasing at other times, except at 08.00 h, when rER volume and surface density were relatively high. The plasma renin activity was maximal at 20.00 h, whereas it was minimal at 08.00 h. The variation in plasma angiotensinogen concentrations was inversely correlated with that in plasma renin activities. These results suggest that JG cells actively synthesize and release renin during the dark period, especially at 20.00 h, whereas during the light period they gradually synthesize renin and produce the granules, most of which may be stored in the cells during this period.     

The age at onset of diabetes influences functional and structural changes in the pituitary-thyroid axis of streptozocin-diabetic male rats

Severe structural changes leading to marked alterations in secretory activity are known to occur in the pituitary-thyroid axis 1 month after induction of postpuberal streptozocin (SZ)-diabetes. However, SZ-diabetic rats of different age groups have not been compared, nor has the maturity of the pituitary and thyroid glands at the onset of diabetes been correlated with the type and evolution of functional and structural changes. We thus induced diabetes in 1-month (prepuberal of 3-month (postpuberal) old male rats and compared diabetic with control groups 4 and 8 months after SZ or saline injection. We determined: 1) pituitary and thyroid weights, 2) the basal plasma TSH, T3, and T4 concentrations, and 3) several morphometrical measurements in the pituitary and thyroid glands. After 4 months, 1) the pituitary and thyroid weights were decreased, 2) plasma TSH and T3 were unchanged, plasma T4 was reduced. and 3) the number of thyrotropes, degenerative changes of follicle cells, and colloid area were increased, the follicle cell height as well as the number of fused cold follicles decreased, and the follicle area was unchanged in diabetic compared with control rats. The lesions were more conspicuous in pre- than in postpuberal diabetic animals. After 8 months, plasma TSH, T3, and T4 were decreased in diabetic compared with control rats. Except for the increased colloid area, all other lesions were similar, though more severe in prepuberal diabetic rats after 8 than 4 months. Few changes were found in postpuberal diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analyses of atrial myoendocrine cells and plasma atrial natriuretic peptides (ANP) of the rat with special reference to the twenty-four-hour variations in secretory granules and plasma ANP concentrations

Summary Subcellular structures of atrial myoendocrine cells in the rat heart and plasma concentrations of atrial natriuretic peptides (ANP) were examined at six evenlyspaced time points over 24 h, using morphometric techniques and radioimmunoassay.Myofibrils and mitochondria of the cells occupied 73.3% of the cytoplasm; 2% of the cytoplasm was occupied by secretory granules, rough endoplasmic reticulum and Golgi complexes, structures characteristic of endocrine cells. Plasma ANP concentration was maximal at 08.00 h, when the individual volume of secretory granules was minimal. The numerical density of secretory granules was increased at 12.00 h. The plasma ANP concentration was minimal at 20.00 h, when the numerical density was minimal and the individual volume was maximal. The fluctuation in plasma ANP concentrations over 24 h was thus parallel to that in the numerical densities of secretory granules and inverse to that in individual volumes.These results suggest that in rats the secretory activity of atrial myoendocrine cells increases at the beginning of the resting period, whereas it decreases at the beginning of the active phase.     

Effects of somatostatin on human thyroid folliculi in vitro

Somatostatin (SRIF) inhibits calcitonin and T3-T4 secretion in thyroid. We have investigated the in vitro effect of SRIF on the basal and TSH induced [3H]thy incorporation, thyroglobulin (tgb) RNA and cAMP level in follicular cells, isolated from normal and adenomatous human thyroids. [3H]thy uptake has been evaluated as TCA-precipitable material in 2, 4, 8, 24 h incubated follicles and 24 h incubated adherent cells. Tgb RNA has been quantified with cytoplasmic dot blot hybridization and cAMP level with RIA method. SRIF reduces basal and TSH-induced [3H]thy in both suspension follicles and epithelial adherent cells. However it does not modify tgb RNA nor cAMP levels in incubated follicles. These data suggest a direct antiproliferative effect of SRIF on human thyroid.     

Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroid-stimulating hormone receptor-adenyl cyclase complex

When cultured in collagen gel-coated dishes, thyroid cells organized into polarized monolayers. The basal poles of the cells were in contact with the collagen gel, whereas the apical surfaces were facing the culture medium. Under these culture conditions, thyroid cells do not concentrate iodide nor respond to acute stimulation by thyroid-stimulating hormone (TSH). To allow the free access of medium components to the basal poles, the gel was detached from the plastic dish and allowed to float in the culture medium. After release of the gel, the iodide concentration and acute response to TSH stimulation were restored. Increased cAMP levels, iodide efflux, and formation of apical pseudopods were observed. When the thyroid cells are cultured on collagen-coated Millipore filters glued to glass rings, the cell layer separates the medium in contact with the apical domain of the plasma membrane (inside the ring) from that bathing the basolateral domain (outside the ring). Iodide present in the basal medium was concentrated in the cells, whereas no transport was observed when iodide was added to the luminal side. Similarly, an acute effect of TSH was observed only when the hormone was added to the basal medium. These results show that the iodide concentration mechanism and the TSH receptor-adenylate cyclase complex are present only on the basolateral domain of thyroid cell plasma membranes.     

Response of the thyroid gland of the lizard Podarcis sicula to endothelin-1

Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET) induced hormonal functions in various tissues. We have studied the interaction of ET-1 with thyroid gland of the lizard Podarcis sicula. The effects of ET-1 administration on the plasma levels of the thyroid hormones (T3 and T4) and TSH were stimulatory. Morphological changes in the thyroid after treatment with ET-1 were also detected: the height of the epithelial cells slightly increased and the apical surface acquired microvilli protruding into the follicular lumen. The colloid filled up the lumen and showed a rich peripheral vacuolation. In conclusion, a modulatory role in the control of the reptilian thyroid gland is suggested for ET-1. This is the first report on the interaction of ET-1 with the thyroid gland of reptilian.     

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells

Summary Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing the intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.     

CHANGES IN FINE STRUCTURE AND ACID PHOSPHATASE LOCALIZATION IN RAT THYROID CELLS FOLLOWING THYROTROPIN ADMINISTRATION

Shortly after the administration of 1/40 unit thyrotropin to rats, 24 hours post-hypophysectomy, the following sequence of changes has been observed within thyroid follicular epithelial cells: (1) the appearance of apical cell surface activity consisting of pseudopods projecting into the follicular lumen; (2) apparent phagocytic engulfment of colloid droplets lacking indications of acid phosphatase activity; (3) close association and probable fusion of newly formed colloid droplets and dense granules, the latter cytochemically positive for acid phosphatase activity; (4) the appearance of presumptive acid phosphatase activity within colloid droplets; and, (5) further colloid droplet changes, viz., basipetal migration and decrease in size, accompanied by an increase in density and in demonstrable acid phosphatase activity. These changes appeared to represent the resorption and degradation of follicular colloid. Comparable results were obtained using intact and more heavily stimulated animals. Colloid biosynthesis was tentatively visualized in these cells as a separate mechanism involving small vesicles prominent in the Golgi region and beneath the apical plasma membrane of some, but not all, thyroid follicular cells in each specimen.     

Distribution of microtubules and microfilaments in thyroid follicular epithelial cells of normal,TSH-treated,aged, and hypophysectomized rats

Summary We investigated the distribution of microtubules and microfilaments in rat thyroid follicular epithelial cells by applying an immunofluorescence technique with monoclonal antibodies against tubulin and by staining sections with rhodamine-phalloidin. In normal thyroid cells, microtubules run longitudinally from the apical region to the basal region intersecting with each other. In addition, intense labelling with tubulin antibodies was observed in the apical part of the cell. The ultrastructural examinations showed that microtubules often run along the apical plasma membrane. Dot-like labelling with anti-tubulin antibodies was often observed in the perinuclear space, but no microtubules were recognized in the nucleus. Microfilaments bound to rhodamine-phalloidin were distributed mainly beneath the apical plasma membrane, and the portion along the basolateral membrane was scarcely positive. The apical pole of the follicle cell was also decorated by anti-microtubule-associated protein-2 (MAP-2). After TSH stimulation, the intensity of immunocytochemical staining against tubulin was remarkably increased in the cytoplasm. Simultaneously, at the apical region, the staining intensity of rhodamine-phalloidin was increased. Microtubules and microfilaments appeared in the pseudopods after TSH injection. In hypophysectomized or aged rats, thyroid follicular epithelial cells decreased in height, and both immunofluorescent labelling against tubulin and rhodamine-phalloidin labelling were markedly decreased. These results indicate that the distribution and polymerization of microtubules and microfilaments in thyroid follicular epithelial cells vary with the functional stage.     

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.     

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.     

Cholesterol-rich microdomains in rat and porcine thyroid membranes involved in TSH-induced endocytotic processes

Filipin, a polyene antibiotic, was used to detect cholesterol in thyroid membranes in vivo and in culture during TSH stimulation. We found that apical and basolateral plasma membranes were heterogeneously modified by filipin which induced abundant lesions in apical membranes, whereas Golgi apparatus, endoplasmic reticulum, nuclear membranes were unmodified. Small apical vesicles and colloid droplets were generally highly enriched in these complexes, suggesting a high cholesterol concentration in their membranes. Pseudopod membranes, known to be highly specialized domains in the apical plasma membrane, appeared enriched in cholesterol. Consequently, we suggest that an increased cholesterol content may be involved in the stabilization of thyroid membranes during endocytotic processes.     

STUDIES ON SMALL INTESTINAL CRYPT EPITHELIUM : I. The Fine Structure of the Crypt Epithelium of the Proximal Small Intestine of Fasting Humans

Small intestinal crypt epithelium obtained from normal fasting humans by peroral biopsy of the mucosa was studied with the electron microscope. Paneth cells were identified at the base of the crypts by their elaborate highly organized endoplasmic reticulum, large secretory granules, and small lysosome-like dense bodies within the cytoplasm. Undifferentiated cells were characterized by smaller cytoplasmic membrane-bounded granules which were presumed to be secretory in nature, a less elaborate endoplasmic reticulum, many unattached ribosomes and, in some cells, the presence of glycogen. Some undifferentiated cells at the base of the crypts contained lobulated nuclei and striking paranuclear accumulations of mitochondria. Membrane-bounded cytoplasmic fragments, probably originating from undifferentiated and Paneth cells, were frequently apparent within crypt lumina. Of the goblet cells, some were seen actively secreting mucus. In these, apical mucus appeared to exude into the crypt lumen between gaps in the microvilli. The membrane formerly surrounding the apical mucus appeared to fuse with and become part of the plasma membrane of the cell, suggesting a merocrine secretory mechanism. Enterochromaffin cells were identified by their location between the basal regions of other crypt cells and by their unique intracytoplasmic granules.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Food restriction induced thyroid changes and their reversal after refeeding in female rats and their pups

In the present study, two groups of pregnant female rats were submitted to food restriction (24 h fast versus 24 h diet intake) from the 14th day of pregnancy until either the 14th day (group B) or the 4th day after parturition (group C). All pups and their mothers were sacrificed on day 14 after delivery. The body weight of the 14-day-old pups (group B) was 46% less than the controls (group A). Free thyroxine and free triiodothyronine levels in the plasma were reduced by 44 and 16% in pups and by 20 and 36% in their mothers, respectively. These reductions were correlated with a decrease in thyroid iodine content of the pups (-50%) and their mothers (-24%). Radioiodine uptake (131I) by the thyroid gland of pups was significantly increased by 27%. Plasma TSH levels were decreased by 38% in pups and by 44% in dams. Morphological changes in thyroid glands were observed in energy restricted dams and in their pups. Some of follicles in pups were empty. Moroever in dams, we noted the presence of peripheral resorbed vacuoles, sign of thyroid hyperactivity. After a refeeding (group C) period of ten days, total recovery occurred in plasma thyroid hormone levels (FT4 and FT3) and in thyroid iodine contents of pups in spite of a partial recovery of body weights and plasma TSH levels. In dams, a partial recovery occurred in plasma thyroid hormone levels in spite of total recovery in thyroid iodine contents, while plasma TSH levels exceeded control values. A significant amelioration in thyroid histological aspects was observed in pups and their dams.