Peroxidase Secretion Research Articles

Hormonal influence on expression and functionality of α 1-adrenoceptor in rat submandibular gland

In this work, we characterized α 1-adrenoceptor expression and functionality in rat submandibular gland. Cumulative dose–response curve of methoxamine was constructed to determine the peroxidase secretion by glands from proestrous, estrous, metestrous and diestrous rats. They were compared to those from animals untreated or treated with sex hormones, estradiol and progesterone. The sensitivity of glands to an α 1-adrenoceptor agonist varied depending on hormonal state, i.e. glands from proestrous and estrous were more sensitive to the stimulatory action of methoxamine than those from metestrous, diestrous and ovariectomised animals. The efficacy of the α 1 agonist was enhanced in glands from ovariectomised estrogen-treated rats but it was ineffective in glands from ovariectomised progesterone-treated rats. The functional studies correlated with 3H-prazosin binding assays in which estrogen increased α 1-adrenoceptor density while progesterone decreased it. The results demonstrated that α 1-adrenoceptor expression and functionality in rat submandibular glands are apparently under hormonal control and probably represent other examples of bidirectional interactions between neuronal and exocrine systems.

Effects of ageing on secretagogue-evoked protein output, peroxidase secretion and protein synthesis in the isolated rat lacrimal gland.

The exorbital lacrimal glands secrete the aqueous component of the tear film that consists mainly of water, electrolytes and proteins.1 Lacrimal protein output and fluid secretion are regulated by the autonomic nerves that innervate the acini, ducts and blood vessels.2 In a previous study we showed the typical serous acini of the lacrimal gland of young (3–5-month-old) male rats contained numerous protein secretory granules. Ageing was associated with a progressive loss of protein (from serous to seromucus and subsequently mucus acini). In addition, marked changes and patchy destruction of the endoplasmic reticulum and Golgi apparatus were observed in the acini of glands in aged rats when compared to that from young rats.3,4 Taken together, these observations suggest ageing may involve a derangement in lacrimal protein synthesis and release. This study investigates differences in secretagogue-evoked protein and peroxidase secretion and protein synthesis in male rat lacrimal acini.KeywordsOcular AllergyVernal KeratoconjunctivitisTopical CyclosporineAtopic KeratoconjunctivitisTolypocladium InflatumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Heterologous expression and secretion of sweet potato peroxidase isoenzyme A1 in recombinant Saccharomyces cerevisiae

A vector system has been developed to express isoenzyme A1 of sweet potato peroxidase (POD) and was introduced into Saccharomyces cerevisiae. The system contains the signal sequence of Aspergillus oryzae α-amylase to facilitate the extracellular secretion of peroxidase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. In a batch culture using YNBDCA medium (yeast nitrogen base without amino acids 6.7 g l −1 , Casamino acids 5 g l −1 and glucose 20 g l −1 ), the recombinant strain expressed the swpa1 gene giving a secretion yield of POD activity of ca. 90% of total expressed peroxidase. Supplementation with PMSF (0.05 mM) and Casamino acids (5 g/50 ml) increased extracellular POD activity to nearly 10 kU ml −1 , equivalent to 1.5 kU g −1 cell dry wt. This is 9 fold higher than that obtained in medium without PMSF. From SDS-PAGE and native-PAGE analyses POD has an Mr of 53 kDa.

Interaction of calcitonin gene-related peptide (CGRP), substance P (SP) and conventional autonomic agonists in rat submandibular salivary peroxidase release in vitro

Our previous immunohistochemical studies reveal that several neuropeptides, such as substance P and calcitonin gene-related peptide, innervate the major salivary glands of the mouse, rat and human. The aim of the study was to clarify their interactions by measuring their effects alone or with conventional autonomic agonists (carbachol, phenylephrine and isoproterenol) on peroxidase secretion of incubated submandibular gland slices. Calcitonin gene-related peptide evoked significant increase in peroxidase activity of the secretion only when used at 10 −5 M concentration, while substance P evoked significant, dose-dependent increase at much lower concentrations (10 −10 M). Adrenergic and cholinergic agonists enhanced peroxidase activity. Interestingly, substance P inhibited both phenylephrine and isoproterenol induced increase in peroxidase activity. Calcitonin gene-related peptide did not affect the inhibition caused by substance P. Our results demonstrate that in the salivary gland tissue substance P alone or in conjunction with adrenergic agonists result in opposing secretory responses with the doses used in vitro. Conversely, the response mediated by adrenergic receptors may be critically affected by simultaneous occupation of substance P receptors.

An analysis of submandibular salivary gland function with desipramine and age in female NIA Fischer 344 rats

Dry mouth is one of the major side effects of cyclic antidepressants, which are still a dominating group of psychotherapeutic drugs used in the treatment of depression. In this study we analyzed the effects of 28 day tricyclic antidepressant administration and the reversibility of this treatment following a 15 day washout period on different parameters in submandibular gland function in aging rats. We postulated that desipramine would decrease gland function, accented with age, and delay recovery in senescent animals. In contrast to body weight, desipramine had no effect on glandular wet weight. While glandular DNA synthesis was changed with age and treatment, the concentration of total membrane and soluble proteins was not affected. Flow rate was significantly changed with age, but desipramine increased salivary flow in the youngest animals only. Neither age nor treatment influenced salivary protein concentrations, but the total amount of proteins secreted, revealed perturbation with age. SDS- polyacrylamide gel analysis revealed changes in protein expression with treatment and age. Desipramine decreased epidermal growth factor (EGF) levels in all age groups, but increased the secretion of peroxidase and lysozyme. Analysis of total RNA showed a pronounced decrease with age. These data indicate that desipramine has profound effects on submandibular salivary gland function.

Immunoglobulin a Secretion into Saliva During Dual Sympathetic and Parasympathetic Nerve Stimulation of Rat Submandibular Glands

Salivary secretion of proteins from rat submandibular glands was studied using graded stimulation of the parasympathetic nerve in isolation, and then at a fixed rate in combination with graded sympathetic nerve stimulation. Increasing the frequency of parasympathetic nerve stimulation per se caused a gradual increase in the secretion of peroxidase (from acini) but only small increases in proteinase (from ductal cells) and IgA outputs. Dual stimulations, with an increasing frequency of sympathetic nerve stimulation on a background of low frequency parasympathetic nerve stimulation, showed that maximal acinar secretion of peroxidase required only a low frequency of additional sympathetic stimulation, whereas ductal secretion of kallikrein was greatest with the highest frequency of additional sympathetic stimulation (20 Hz in bursts). IgA secretion also required high frequency additional sympathetic stimulation in bursts for greatest output. Although a synergism occurred with parasympathetic plus sympathetic nerve stimulation for the secretion of both peroxidase and kallikrein it was not evident for the secretion of IgA. This presumably reflects a difference for exocytosis of proteins stored in granules (e.g. peroxidase and kallikrein) compared to those proteins continuously transported across the plasma membrane in vesicles by transcytosis. This work confirms that vesicular movement of secretory IgA can be increased by both parasympathetic and sympathetic nerve stimulation, but the frequency parameters differ for each nerve.

Targeting of aminopeptidase N to bile canaliculi correlates with secretory activities of the developing canalicular domain.

We have used human hepatoma cell lines as an in vitro model to study the development of hepatic bile canaliculi (BC). Well-differentiated hepatoma cells cultured for 72 hours could develop characteristic spheroid structures at sites of cell-cell contact that contained tight junctions and various membrane protein markers, resembling BC found in vivo. Intact cytoskeleton was essential for this differentiation process. In the coculture experiments in which cells of different origins were populated together, BC only formed between hepatic cells and preferentially among well-differentiated cells. Poorly differentiated hepatoma cells never formed BC among themselves, but could be induced to undergo canalicular differentiation by interacting with well-differentiated cells. During BC morphogenesis, integral canalicular membrane proteins were gradually delivered and accumulated at the developing BC. Among them, targeting of aminopeptidase N (APN) seemed to correlate with activation of certain secretory functions. Specifically, only APN-positive BC supported excretion of fluorescein diacetate (FDA) and 70-kd dextran, but had no relationship with secretion of horseradish peroxidase (HRP). Targeting of another BC protein, dipeptidyl peptidase IV (DPPIV), on the other hand, bore no association with any secretory activity examined. In addition, inhibition of enzymatic activity of APN could perturb canalicular differentiation without affecting cell proliferation. Our results suggest that targeting of APN proteins may reflect or even play an important role in the development and functional maturation of the canalicular structures.

Glycosylation and its Adequate Processing is Critical for Protein Secretion in Tobacco BY2 Cells

Peroxidases are secreted into the culture medium of BY2 suspension cells. In order to determine the role of glycosylation and glycan processing for peroxidase secretion, inhibition studies were performed. When glycosylation was inhibited by tunicamycin, secretion of peroxidases into the medium ceased totally. When glycan processing was inhibited by castanospermine, the molecular size of the peroxidases was increased concomitant with their decreased secretion into the medium. Upon inhibition treatment, pulse-labeled total secreted protein behaved similarly to the peroxidases, indicating that for secretion, glycosylation is generally essential, while glycan processing is not prerequisite but none the less necessary. These studies are supported by the observation that inhibitor treatment also induced transcript accumulation for the molecular chaperon, BiP, and for calnexin, which recognizes immature glycans. Taken together, these results strongly suggest that correct N-glycan structure is critical for protein secretion in plant cells.

Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells.

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell Physiol. 39): C1743-C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the beta1 and gamma1 chains, but not alpha1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the beta1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.

98/01877 A test system to analyse the complex physiological states of coal solubilizing fungi : Holker, U. et al. Fuel Process. Technol., 1997, 52, (1–3), 65–71

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN3, ascorbic acid, β-mercaptoethanol and dithreitol. The Km values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.

Effects of neuropeptides on serotonin release and protein and peroxidase secretion in the isolated rat lacrimal gland.

The secretion of tears by the lacrimal gland is regulated by the autonomic nervous system1 and by non-cholinergic, non-adrenergic (NCNA) nerves that are believed to release a number of biologically active peptides that can function as neurotransmitters of NCNA nerves regulating the secretory processes.2,3 The neuropeptides have been investigated for their potential role in modulating the immune system. In many organs and tissues from several animal species, a morphological and functional association was found between mast cells and neuropeptide-containing nerves that contain substance P (Sub P) and calcitonin gene-related peptide (CGRP), or both.4,5 Mast cells have a secretory capacity. They released mediators which include serotonin (5-HT), and histamine which can evoke pronounced biological effects resulting in cellular regulation.6 It is now apparent that neuropeptides can modulate some immune response directly,7 and several peptides--in particular, Sub P have been shown to induce mast cell mediator release.8 It is possible that neuropeptides may function as immunological effectors in inflammatory responses in the lacrimal gland. Moreover, in a previous study we showed that the lacrimal gland is innervated with numerous neuropeptides, and there is a consistent association between peptidergic nerves and mast cells.9 This study was designed to investigate any functional interaction between mast cells and peptidergic neurotransmitters Sub P and CGRP.KeywordsMast CellAcinar CellLacrimal GlandSodium CromoglycateSerotonin ReleaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Protein Secretion from Rat Submandibular Acini and Granular Ducts: Effects of Exogenous VIP and Substance P During Parasympathetic Nerve Stimulation

The influences of exogenous vasoactive intestinal peptide (VIP) and substance P on the release of peroxidase from acini and true tissue kallikrein (rK1) from granular ducts of the rat submandibular gland were studied during continuous parasympathetic stimulation. Parasympathetic nerve impulses caused a moderate flow of saliva (mean ± SD, 108 ± 26 μl/g tissue/min) that had a low protein concentration (174 ± 88 μg/ml). The outputs of peroxidase and rK1 were minimal (14.3 ± 11.8 pmol DCF/g tissue/min and 6.5 ± 3.4 nmol AFC/g tissue/min, respectively). When administered intravenously, VIP had no apparent effect on the overall flow rate, but caused a significant increase in the output of peroxidase; 450% at 1 μg/kg and a further 10-fold increase at 10 μg/kg. In contrast, substance P (1 μg/kg) evoked a marked increase in flow rate (68%), and peroxidase secretion increased only 3-fold. The output of rK1 was unaffected by either VIP or substance P. Our results support the hypothesis that acinar, but not granular duct, protein secretion is evoked by non-adrenergic, non-cholinergic peptides released from parasympathetic nerve terminals.

Secretory interactions between the sympathetic and parasympathetic innervations of the submandibular gland in the anaesthetized cat.

Interactions between the sympathetic and parasympathetic innervations of the submandibular gland have been investigated in the anaesthetized cat. At low frequencies of chorda lingual (parasympathetic) stimulation, simultaneous stimulation of the ascending cervical sympathetic nerve in bursts (20 Hz for 1 s at 10 s intervals) increased the flow of submandibular saliva, but the effect was never more than additive. The output of protein was consistently reduced by simultaneous stimulation of both the sympathetic and parasympathetic innervations, below that evoked by stimulation of either alone. Sympathetic stimulation was more effective than parasympathetic stimulation in promoting the secretion of tissue kallikrein and peroxidase in the submandibular saliva. The output of the latter enzyme, in response to sympathetic stimulation, was significantly reduced by simultaneous stimulation of the parasympathetic innervation at frequencies greater than 1 Hz, but nevertheless exceeded the amount secreted during chorda stimulation alone. Thus, this protocol provided no evidence of synergy between the two divisions of the autonomic nervous system with respect to any submandibular secretory function that was recorded. However, following the administration of a small dose of atropine (2-15 micrograms kg-1 i.v.), sufficient to block secretion during chorda stimulation alone, the flow of saliva, in response to sympathetic stimulation, was potentiated when superimposed on a background of parasympathetic stimulation at all frequencies that were employed. This effect was abolished by larger doses of atropine, indicating that it was dependent upon activation of muscarinic receptors, only some of which could have been blocked by the initial dose.

Control of porcine lacrimal gland secretion by non-cholinergic, non-adrenergic nerves: effects of electrical field stimulation, VIP and NPY

This study employs the technique of electrical field stimulation (EFS) to characterise the effects of endogenous neurotransmitters on protein secretion in the in vitro pig lacrimal gland. The effects of exogenous applications of neurotransmitters on protein output and peroxidase secretion were also investigated for comparative purposes. EFS evoked frequency-dependent (5–20 Hz) increases in protein secretion. The EFS-evoked protein output was abolished with the nerve blocking drug tetrodotoxin (10 −6 M, TTX). Elevated potassium (100 mM KCl) can stimulate protein output in the presence of TTX. Exogenous application of either acetylcholine (ACh. 10 −9−10 −4 M) or noradrenaline (NA, 10 −8−10 −4 M) can also result in protein secretion, but they have no detectable effect on peroxidase secretion. In the presence of the cholinergic antagonist, atropine (10 −5 M) the EFS-induced protein output was reduced but not abolished. This atropine-resistant and non-cholinergic nerve-mediated component was further reduced in the combined presence of atropine, phentolamine, and propranolol (all 10 −5 M). When vasoactive intestinal polypeptide (VIP) receptor antagonist (10 −6 M[4-Cl- d-Phe 6-Leu 17]-VIP) was combined with the cholinergic and adrenergic antagonists, EFS caused a small but detectable increase in protein output. Exogenous application of either 10 −9 M VIP or 10 −9 M neuropeptide-Y (NPY) resulted in protein secretion. Combination of both VIP and NPY only induced an additive effect on protein output. Theophylline (10 −4 M), a phosphodiesterase inhibitor, evoked a small increase in protein output and had no significant effect on the secretory responses elicited by either VIP or NPY. In contrast, theophylline potentiated the non-cholinergic, non-adrenergic EFS-induced protein secretion. The results indicate that protein secretion from the porcine lacrimal gland may be controlled by cholinergic, adrenergic and non-cholinergic, non-adrenergic nerves. The peptidergic neurotransmitters may be VIP and other related neuropeptide(s). In addition to these neurophysiological studies, our results confirm previous findings that the porcine lacrimal nerves contain abundant quantity of NPY and VIP.

Effect of Enantiomeric Forms of Albuterol on Stimulated Secretion of Granular Protein from Human Eosinophils

We studied the effect of R-, S- and R,S-albuterol in inhibiting the eosinophil peroxidase (EPO) secretion caused by 10−10to 10−6mformyl-met-leu-phe+5μg/ml cytochalasin B (FMLP/CB) in non-allergic and allergic subjects. Total RAST score obtained for allergic subjects was 4.12±0.21 vs 0.36±0.17 for non-allergic subjects (P<0.0001). Stimulated EPO secretion was comparable in allergic [2,051±567 ng/106eosinophils (eos)] and non-allergic subjects [2,337±488 ng/106eos (P=NS)]. At all concentrations used, both R- and R,S-enantiomers caused comparable (27–32%) inhibition of FMLP/CB stimulated secretion of EPO in allergic and non-allergic subjects. Pretreatment with S-albuterol caused no augmentation of EPO secretion in either allergic (115±34.6%) or non-allergic subjects (114±23.7%) subjects, and there was no significant difference in secretion caused by FMLP/CB alone in either experimental group. Similar results were obtained for subjects stratified according to serum IgE concentration. Our data demonstrate that both R- and R,S-albuterol are equivalently effective in inhibiting stimulated secretion of EPO in both normal and allergic subjects and that there is no paradoxical augmenting effect of S-albuterol in stimulated eosinophil secretion.

Enhanced secretion of peroxidase from carrot hairy roots using polyethylene glycol

Effects of various organic solvents and polyethylene glycol (PEG) on the secretion of peroxidase from carrot hairy roots were investigated. The peroxidase activity in culture broth was significantly enhanced without adverse effects on root growth when PEG was employed while other organic permeabilizing agents exerted harmful effects on root growth. PEG with a molecular weight of over 6,000 g/mol enhanced the secretion of peroxidase dramatically. Since PEG did not induce the formation of pores in the root cell membrane as confirmed by staining using Neutral Red, it was thought that the mechanism of secretion enhancement of peroxidase by PEG is different from that of other organic solvents which make the cell membrane permeable. Since PEG does not adversely affect the root growth, PEG can be applied to the repeated use of root cells for production of peroxidase.

Effects of Cholestatic Agents on the Structure and Function of Bile Canaliculi in Neonatal Rat Hepatocytes in Primary Culture.

The effects of cytochalasin B and colchicine on the structure and function of bile canaliculi were studied in neonatal rat hepatocytes in primary culture. Cellular contacts of neonatal hepatocytes were not as tight as those of adult hepatocytes. There was no remarkable difference in the ultrastructure of bile canaliculi between neonatal and adult hepatocytes. Neonatal hepatocytes treated with cytochalasin B were round in shape and aggregated in groups of several cells. Actin filaments stained by rhodamine-phalloidin were disrupted and condensed at the cell periphery or around dilated bile canaliculi. Markedly-dilated bile canaliculi with less microvilli were observed by transmission electron microscopy while the secretory function of horseradish peroxidase, which was used as a marker for uptake, transport and secretion into bile canaliculi, were maintained. The lumen of dilated bile canaliculi was found close to the undersurfaces of hepatocytes by scanning electron microscopy after turning over the cultured cells. By colchicine treatment, the filamentous structure of microtubules in neonatal hepatocytes disappeared. The ultrastructure of the bile canaliculi was not affected by the treatment, but transport and secretion of horseradish peroxidase into bile canaliculi were inhibited. The development of strict cellular polarity in neonatal hepatocytes may be suppressed in neonatal hepatocytes; however, cholestatic agents which rearrange the cytoskeleton caused the same morphological or functional changes of bile canaliculi as in adult hepatocytes.

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation

Sequential secretion of rat submandibular kallikrein and peroxidase during intermittent sympathetic stimulation

Secretion of peroxidase from acinar cells, true tissue kallikrein (rKI) from granular tubules and total protein have been assessed in sequential samples of rat submandibular saliva formed during intermittent periods of sympathetic stimulation in bursts of 50 Hz 1 s every 10 s at 5 V, for 1 or 2 min each with 2 min intervals between periods. This protocol was repeated twice after 1 h rest pauses. The salivary flow remained greater throughout than when using ongoing burst stimulation. Protein secretion was considerable in the first 2 samples (1 min followed by 2 min) reaching the remarkable concentration of 285 ± 14.4 mg/ml, then it gradually decreased with little recovery after 1 h pauses. rKl outputs followed a similar pattern. Peroxidase, however, showed no greater output in initial samples and continued steadily in similar amounts throughout. When intermittent stimulations were used for 1 sequence only (total stimulation time = 9 min) the glands showed 84 ± 2.6% depletion of rK1 compared to the control glands. A correspondingly large depletion of granules occurred from the granular tubules on the stimulated side, which was greater than with ongoing burst stimulation for 1 h (Garrett et al., 1991). Thus, secretion of rK1 from the granules in granular tubules occurs most efficiently with short sharp bursts of high frequency sympathetic stimulation but soon diminishes. In contrast, peroxidase secretion from acinar cells continues steadily and more modestly for long periods of time undiminished.

Secretion and serotonin release in the isolated rat lacrimal gland: the effects of substance P and calcitonin gene-related peptide

A close anatomical relationship between nerves containing substance P and calcitonin gene-related peptide (CGRP) and mast cells containing serotonin has been demonstrated in the rat lacrimal gland. This study investigates the potential for peptidergic regulation of lacrimal mast cells by examining the actions of substance P, CGRP and serotonin on protein and peroxidase secretion from isolated lacrimal segments and on substance P and CGRP to release serotonin from the lacrimal mast cells. substance P, CGRP and serotonin evoked marked increases in total protein and peroxidase from the lacrimal. sodium cromoglycate, a mast cell stabilizer, significantly reduced or blocked the secretory responses elicited by these agonists. Chromatographic analysis using electrochemical detection revealed that substance P, but not CGRP, augmented the release of serotonin from the gland. The substance P evoked peroxidase secretion and serotonin release was blocked by CGRP and by sodium cromoglycate. These results support a role for mast cells in the regulation of lacrimal secretion and suggest a novel regulatory interaction between substance P and CGRP in the control of lacrimal function through a neuro-immune interaction.