Quantification of cysteinyl sulfhydryl residues in peptides and proteins by ESI-MS or MALDI-MS.
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Cysteine↗
Biomedical subjects
G Hellekant
Publications and source records attributed to G Hellekant.
Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B.
We have discovered a new high-potency thermostable sweet protein, which we name brazzein, in a wild African plant Pentadiplandra brazzeana Baillon. Brazzein is 2,000 times sweeter than sucrose in comparison to 2% sucrose aqueous solution and 500 times in comparison to 10% of the sugar. Its taste is more similar to sucrose than that of thaumatin. Its sweetness is not destroyed by 80 degrees C for 4 h. Brazzein is comprised of 54 amino acid residues, corresponding to a molecular mass of 6,473 Da.
Amino Acid Sequence↗
Enhancing effects of saccharin on gustatory responses to D-phenylalanine in monkey single chorda tympani fibers.
Taste enhancing effects of sodium saccharin (Sac) on D-phenylalanine (D-Phe), first found in mice, were examined by comparing single fiber responses to various taste stimuli in the monkey chorda tympani nerve. Fifteen fibers sampled were divided into the following 5 groups according to their responsiveness to 5 prototypical taste stimuli; 8 sucrose-, 2 quinine-, 2 acid-, 2 NaCl- and one monosodium glutamate (MSG)-best fibers. Out of 8 sucrose-best fibers, 5 fibers showed enhancement of D-Phe responses after the stimulation with Sac, but neither the remaining 3 sucrose-best fibers nor other fibers showed the enhancement. These results suggest that (1) the enhancement of D-Phe responses by Sac also occurs in the monkey peripheral taste system, and (2) there exist distinct receptor sites for D-Phe responsible for occurrence of the enhancement, and (3) taste cells possessing the D-Phe receptor site are innervated by a limited subpopulation of sucrose-best fibers.
Animals↗
Sweet taste in the calf: III. Behavioral responses to sweeteners.
The hedonic response to the sweeteners acesulfame-K, aspartame, fructose, galactose, glucose, glycine, lactose, maltose, Na-saccharine, sucrose, and xylitol was recorded in five groups of 4-16-week-old calves. The compounds were presented to the calves for 12 or 24 h in two-bottle preference tests with tap water as one choice. Glycine (10 mM and higher), sucrose (20 mM and higher), and fructose concentrations were most preferred. Sodium-saccharine was highly preferred at and above 4 mM concentration, fructose and lactose were preferred above 40 mM, galactose was preferred moderately, acesulfame-K and maltose were preferred inconsistently, and aspartame and xylitol were not preferred at any concentration. The change of preference during the tests was also studied. Three types of consumption changes were observed. 1) Increased preference of the tastant during consumption, seen during sucrose and, to lesser a extent, fructose consumption. 2) Initial high preference for the tastants, diminishing during the test period, observed with fructose, galactose, glucose, glycine, lactose, and maltose. 3) Initial large fluctuations in consumption from the two bottles, but no change in overall preference. This pattern was seen with xylitol and aspartame. This technique seems to offer a method to assess the long-term preference for a compound within one relatively short two-bottle preference session.
Age Factors↗
Bitter taste in single chorda tympani taste fibers from chimpanzee.
We have found earlier that chimpanzee chorda tympani taste fibers fall into groups that conform with the human taste qualities. This study focuses on bitter taste and its relation to sweet taste. Eight fibers were classified as bitter fibers according to their responses to 31 stimuli. The stimuli included the bitter compounds quinine, denatonium benzoate and caffeine. The results indicate a clear dichotomy between the bitter and sweet fibers. Sweet fibers never responded to the bitter compounds. However, in addition to their responses to the above compounds, some of the bitter fibers were stimulated by other compounds. Most prominent were responses to NaCl-amiloride mixture, KCl and xylitol. In most cases the cause could be assumed to be a bitter taste in the compound. These results suggest that the bitter and sweet tastes are conveyed in specific and separate groups of nerve fibers in the chimpanzee. Because of the closeness between chimpanzee and human, this finding has implications on the question of taste coding in human and the concept of taste qualities.
Animals↗
Ultrastructural evidence for a binding substance to the sweet-tasting protein thaumatin inside taste bud pores of rhesus monkey foliate papillae.
Thaumatin is a protein that tastes intensely sweet only to Old World monkeys and to higher primates, including man. Here we used pre-embedding ultrastructural methods to study the distribution of thaumatin in apical regions of Rhesus monkey foliate papillae, using thaumatin conjugated to 5 nm gold particles. With freeze-substitution we saw that gold-labeled thaumatin bound to an electron-opaque, sponge-like secretory substance inside the taste bud pores. Labeled thaumatin was found at the surface of the secretory substance even deep inside the pore, where other, unlabeled cellular structures surrounded the substance. With freeze-fracture deep-etching the secretory substance that bound the thaumatin-gold particles appeared coarsely granular. There was no labeling of any other taste bud pore structure, including microvilli and small membrane-lined vesicles. Pre-incubation with an excess of unlabeled thaumatin inhibited binding with gold-labeled thaumatin. The results suggest that the secretory substance had the greatest affinity of all taste pore structures to the sweet-tasting compound under our experimental conditions. Therefore, gustatory reception probably involves various taste compound binding structures, microvilli, and also secretory substances like the one described here which bound thaumatin. We speculate that the secretory substance may bind taste stimuli and serve as an intermediate between stimuli and receptors. It could be involved in stimulus removal or delivery or both.
Animals↗
On the taste of umami in chimpanzee.
Whole and single fiber chorda tympani nerve recordings were obtained in 5 chimpanzees to stimulation with MSG (monosodium phosphate) and GMP (guanosine 5'-monophosphate, disodium salt) alone and in combination. The overall chorda tympani nerve activity was recorded to 5 concentrations of MSG, ranging from 1 to 100 mM with and without 0.3 mM GMP, and to 5 concentrations of GMP, ranging from 0.1 to 10 mM, with and without 30 mM MSG. A synergistic effect was recorded between MSG and GMP in 3 out of 4 animals. The effect of stimulation with MSG and GMP alone and mixed was studied in approximately 25 single fiber recordings against a background of the stimulating effects of 11 different sweeteners, 3 acids, 3 bitter compounds and 3 different salts. The fibers showed a high taste specificity and fell into groups which corroborated with the human concepts of the taste qualities. The umami compounds elicited moderate responses which were largest in the sweet fibers. In the 6 sweet fibers that responded to the umami compounds. 0.3 mM GMP was a more effective stimulus than 10 mM MSG. In 3 of these fibers a synergistic effect was recorded to the mixture of GMP and MSG. It is interesting that the response to GMP and MSG was unaffected by gymnemic acid, although it blocked the response to the sweet compounds. Three out of 10 salt fibers responded to MSG and GMP but no synergistic effect was recorded. No specific umami fibers were recorded. However, more data must be collected before the final conclusion on the presence or absence of specific umami fibers can be drawn.
Animals↗
mRNAs for PRPs, statherin, and histatins in von Ebner's gland tissues.
A search was made for expression of genes for proline-rich proteins (PRPs) and other salivary-type proteins, including statherin and histatins, in taste-bud tissues of mice and primates because of previous genetic findings in mice (Azen et al., 1986) that Prp and taste genes for certain bitter substances are either the same or closely linked. Taste-bud tissues and other tissues were tested for specific mRNAs with labeled DNA probes by Northern blotting and in situ hybridization. It was found that PRP mRNAs were present in von Ebner's glands of mice and macaques, and that there was a much greater degree of PRP mRNA induction in mouse parotid (16-fold) than in von Ebner's gland (two-fold) after in vivo isoproterenol stimulation. This difference may be due, in part, to differences in autonomic nerve innervation. Statherin and histatin mRNAs were found in macaque taste-bud tissues containing von Ebner's gland, and statherin protein was found in human von Ebner's gland by immunohistochemistry. The finding of PRP gene expression in von Ebner's gland, whose secretions have been suggested to play a role in taste stimulation, adds further support to a possible function of PRPs in bitter tasting. The possible functions of statherin and histatins in von Ebner's gland secretions may be related to statherin's regulation of salivary calcium and histatins' antibacterial and antifungal properties.
Animals↗
The sweet taste in the calf. I. Chorda tympani proper nerve responses to taste stimulation of the tongue.
Electrophysiological recordings were obtained from the chorda tympani nerve in calves during stimulation with NaCl, quinine hydrochloride, citric acid, acesulfan-K, aspartame, fructose, galactose, glucose, glycine, lactose, maltose, monellin, Na-saccharin, sucrose, thaumatin, and xylitol. In cattle the chorda tympani innervates the posterior third of the tongue as well as the anterior part. It was found that the posterior receptive field generally responded better to sweet substances than the anterior. Glycine and Na-saccharin followed by xylitol were the most effective sweet stimuli. The monosaccharides elicited larger responses than the disaccharides. Aspartame gave a weak nerve response in 5 of 13 calves. Monellin and thaumatin elicited no change in chorda tympani nerve activity and did not crossadapt with any sweetener. No effects on citric acid responses were observed after application of miraculin.
Animals↗
The sweet taste in the calf. II. Glossopharyngeal nerve responses to taste stimulation of the tongue.
Recordings were obtained from the glossopharyngeal nerve in 1-5-week-old calves during stimulation of the circumvallate tongue area with NaCl, quinine hydrochloride, citric acid, and the sweet compounds: acesulfam-K, aspartame, fructose, galactose, glucose, glycine, lactose, maltose, monellin, Na-saccharin, sucrose, thaumatin, and xylitol. All compounds except aspartame, monellin and thaumatin gave a nerve response. Glycine, followed by Na-saccharin, elicited the largest responses. Sucrose gave the largest response among the disaccharides, while there was no significant difference between the monosaccharides. Expressed as percent of the NaCl responses, the responses to glycine, sucrose, xylitol, fructose, galactose, glucose, lactose and maltose were considerably larger in the glossopharyngeal nerve than in the chorda tympani nerve. This can be taken as an indication that the posterior region of the tongue serves as the major receptive area for sweet in cattle.
Animals↗
Evidence for a novel mechanism of binding and release of stimuli in the primate taste bud.
In previous work, we showed that thaumatin, an intensely sweet protein, binds to certain formed elements in taste pores of Rhesus monkey foliate papillae, namely, microvilli and small vesicles shed from microvilli, in addition to amorphous secretions (Farbman et al., 1987). We suggested that the taste bud responds to a thaumatin stimulus by shedding the small vesicles containing fragments of microvillar membrane bearing the stimulus-binding site complex. To examine this hypothesis further, we used electron microscopy to examine taste pores of both vallate and foliate papillae from Rhesus monkeys before or after stimulation with thaumatin or sucrose. We also recorded the neural activity from the glossopharyngeal nerve during stimulation with thaumatin, sucrose, citric acid, and NaCl. The results indicate (1) with no stimulation, vesicles are found in pores of foliate papilla taste buds much more frequently than in pores of vallate papilla buds, (2) in both types of papillae, stimulation with sucrose has no apparent effect on the number of pores containing vesicles, (3) stimulation with thaumatin elicits release of vesicles into pores of both foliate and vallate buds, (4) repeated stimulation of taste buds with thaumatin results in a declining neural response, not seen after repeated stimulation with sucrose, citric acid, or NaCl, and (5) stimulation with thaumatin suppresses the neural response to sucrose, but the reverse does not occur. The combined morphological and physiological data support our original hypothesis that, in response to thaumatin stimulation, binding sites on taste microvillar membranes may be shed as a stimulus-receptor complex into the pore. Alternatively, the binding sites may in some way be altered by the shedding of part of the membrane. The data suggest further that the binding site for sucrose may be close to that for thaumatin because it too is lost or altered after thaumatin stimulation. The reduced neural response after repeated thaumatin stimulation indicates that cells may have suffered a net loss of functional receptors or at least a loss in functional capacity of the receptors. The data indicate that the cellular mechanism for handling the protein sweetener, thaumatin, is strikingly different from the adsorption-desorption response to saccharide sweeteners.
Adaptation, Physiological↗
Labeling of sweet taste binding sites using a colloidal gold-labeled sweet protein, thaumatin.
Thaumatin, an intensely sweet tasting protein, was bound to colloidal gold and applied to the taste bud-bearing foliate papillae of Rhesus monkeys. Examination of thin sections of taste pores showed that gold particles were bound to merocrine secretions of Type I taste bud cells, to some cell remnants of lysed cells, and, most importantly, to small, membrane bounded blebs of cytoplasm. These blebs are thought to be shed into the pore from the tips of taste bud cell microvilli, particularly those arising from Type II cells. The binding of gold particles to microvillus tips and to the blebs suggest that this may be an important means by which taste bud cells rid themselves of taste stimulus-receptor complexes.
Animals↗
Structure of taste buds in foliate papillae of the rhesus monkey, Macaca mulatta.
Taste buds in foliate papillae of the rhesus monkey were examined by electron microscopy. Three distinct cell types were identified. Type I cells were narrow elongated cells containing an oval nucleus, bundles of intermediate filaments, several Golgi bodies, and characteristic apical membrane-bounded dense granules. These cells exhibited morphological variations: some had a moderately dense cytoplasm, perinuclear free ribosomes, and flattened sacs of rough endoplasmic reticulum; others had a more lucent cytoplasm, dilated irregular rough endoplasmic reticulum, lysosome-like dense bodies, and lipid droplets. Type II cells typically contained a spherical, pale nucleus, a prominent nucleolus, supranuclear and infranuclear Golgi bodies, mitochondria with tubular cristae, and one or two centrioles. This cell type, too, showed some variation in the relative amounts of ribosomes and smooth endoplasmic reticulum, which varied inversely with each other. Type III cells were characterized by a clear apical cytoplasm essentially devoid of ribosomes and containing microtubules. In a few type III cells, the peri- and infranuclear regions contained many ribosomes and some rough endoplasmic reticulum. In most Type III cells, there were large numbers of dense and clear vesicles in the peri- and infranuclear regions; some of the vesicles were grouped in synapse-like arrangements with adjacent nerves. The morphological variations exhibited by all three cell types could be accounted for by age differences in each of the cells. This would be consistent with the notion that cell renewal occurs in each of the three cell populations.
Animals↗
Effects of gymnemic acid on the chorda tympani proper nerve responses to sweet, sour, salty and bitter taste stimuli in the chimpanzee.
In man gymnemic acid is able to abolish the sweet taste. Also in man, the neural correlate of that effect is a disappearance of the response to sweet stimuli in the taste nerves, as indicated by the observations of Diamant et al. (1965). Although a variety of other mammals also show neural responses to sweet-tasting compounds, the corresponding effect of gymnemic acid has not been demonstrated. This study presents chorda tympani proper nerve recordings from the chimpanzee before and after gymnemic acid. On the chimpanzee tongue, application of 2 ml gymnemic acid (3-10 mg X ml-1 for 3-4 min) completely abolished the taste responses to 0.0035 M acesulfam-K, 0.0018 M aspartame, 0.015 M D-tryptophan, 0.02% monellin, and 0.02% thaumatin, reduced by 75% the response to 0.3 M sucrose, and by 50% that of 0.76 M xylitol. No decrease was recorded in the responses to 0.001 M quinine, 0.1 M NaCl, 0.02 and 0.04 M ascorbic acid, 0.02 and 0.04 M citric acid. The response to the sweeteners recovered with time and the recovery was complete or nearly complete after one and a half hours. It was also found that after application of 2 ml miraculin, 3 mg X ml-1 for 3 min to the tongue the neural response to acids was about 1.5 times as large as before. Gymnemic acid applied before miraculin prevented this enhancement and gymnemic acid after miraculin depressed the enhancement by miraculin of the response to citric and ascorbic acid.
Animals↗
The sweetness-inducing effect of miraculin; behavioural and neurophysiological experiments in the rhesus monkey Macaca mulatta.
1. The gustatory effects of miraculin, the sweetness-inducing protein from the miracle fruit Synsepalum dulcificum, was studied in the rhesus monkey, Macaca mulatta.2. The intake of five acids was recorded in two-bottle preference tests, one bottle containing acid and the other tap water, before and after miraculin treatment. All the acids tasted more pleasant after miraculin.3. The electrical activity of the chorda tympani nerve to stimulation of the tongue with a variety of sweeteners, acids, sodium chloride and quinine hydrochloride was recorded in anaesthetized animals.4. Pre-treatment of the tongue with 0.3-5 mg miraculin doubled the summated nerve response to the acids and diminished the response to sucrose by about 10%. The enhancement lasted for at least an hour and the diminution up to 20 min.5. After miraculin treatment the Spearman's rank correlation coefficient between the order of increased intake of acids and the order of enhancement of the summated nerve response was 0.99.6. A solution of 0.1 mg miraculin per ml. elicited a weak nerve response. No preference over water for this concentration of miraculin was recorded in the two-bottle tests.7. The activity of twenty-nine single taste fibres, selected for their responsiveness to sweetness or acids or both, was recorded after miraculin treatment. Effects were obtained in nine fibres which were similar but more pronounced than those observed in the summated recordings. Before miraculin, these fibres responded better and to a larger variety of sweeteners (81%) than the other fibres (40%). After miraculin, acids elicited on the average 2.3 times more activity than before, while the response to sweeteners was depressed. In twenty fibres no effect of miraculin was observed. These fibres responded to fewer of the sweeteners and were more stimulated by the non-sweet stimuli than the first group.8. The results suggest that miraculin acts on those structures in the taste cell membrane that are involved in perception of the sweet taste, making them sensitive to acids. The new quality of sweetness after miraculin treatment is signalled by taste fibres which normally respond to sweet substances but which, under the influence of miraculin, are responding to acids. It is likely that the quality of a taste stimulus is conveyed by the identity of the taste fibres.
Acids↗
Neural (electro-physiological) methods in chemoreception research.
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Animals↗
Reference for sweet taste and the taste of sweeteners in animals.
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Animal Feed↗
Decline and disappearance of taste response after interruption of the chorda tympani proper nerve of the rat.
The response amplitude in the chorda tympani proper nerve of rats to taste stimulation has been studied at different times after interruption of the nerve in the middle ear. The results show that the reponse in the nerve declined and disappeared completely within 15 h after the nerve was interrupted. As a first sign of deteriorating function a stage of hypersensitivity was observed. Results obtained during applications of local anesthetic or colchicine and section at different distances from the tongue indicate that the decreased response was the result of an interruption of axoplasmic flow from the nerve cell bodies in the geniculate ganglion to the taste buds.
Anesthesia, General↗