Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage.
Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter of A. awamori or with the gpdA promoter of Aspergillus nidulans. There was good correlation of tha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems in A. awamori that result in very high levels of thaumatin production. (+info)
Taste qualities of solutions preferred by hamsters.
Molecules of diverse chemical structure are sweet to humans and several lines of evidence (genetic, physiological, behavioral) suggest that there may be distinct sweet perceptual qualities. To address how many perceptual categories these molecules elicit in hamsters (Mesocricetus auratus), we studied patterns of generalization of conditioned taste aversions for seven sweeteners: 100 mM sucrose, 320 mM maltose, 32 mM D-phenylalanine, 3.2 mM sodium saccharin, 16 mM calcium cyclamate, 10 mM dulcin and 32 mM sodium m-nitrobenzene sulfonate. Each stimulus was preferred versus water in two-bottle intake tests and stimulated the chorda tympani nerve. For each of seven experimental groups the conditional stimulus (CS) was a sweetener and for the control group the CS was water. Apomorphine.HCl was injected i.p. after a CS was sampled and, after recovery, test stimuli (TS) were presented for 1 h daily. The intake (ml) of each TS consumed by experimental animals was compared with mean TS intake by the control group. Learned aversions for 18/21 stimulus pairs cross-generalized, resulting in a single cluster of generalization patterns for the seven stimuli. Cross-generalization failures (maltose-cyclamate, maltose-sucrose, cyclamate-NaNBS) may be the consequence of particular stimulus features (e.g. salience, cation taste), rather than the absence of a 'sucrose-like' quality. The results are consistent with a single hamster perceptual quality for a diverse set of chemical structures that are sweet to humans. (+info)
Sweet taste responses of mouse chorda tympani neurons: existence of gurmarin-sensitive and -insensitive receptor components.
Inhibitory effects of gurmarin (gur) on responses to sucrose and other sweeteners of single fibers of the chorda tympani nerve in C57BL mice were examined. Of 30 single fibers that strongly responded to 0. 5 M sucrose but were not or to lesser extent responsive to 0.1 M NaCl, 0.01 M HCl, and 0.02 M quinine HCl (sucrose-best fibers), 16 fibers showed large suppression of responses to sucrose and other sweeteners by lingual treatment with 4.8 microM (approximately 20 microg/ml) gur (suppressed to 4-52% of control: gur-sensitive fibers), whereas the remaining 14 fibers showed no such gur inhibition (77-106% of control: gur-insensitive fibers). In gur-sensitive fibers, responses to sucrose inhibited by gur recovered to approximately 70% of control responses after rinsing the tongue with 15 mM beta-cyclodextrin and were almost abolished by further treatment with 2% pronase. In gur-insensitive fibers, sucrose responses were not inhibited by gur, but were largely suppressed by pronase. These results suggest existence of two different receptor components for sweeteners with different susceptibilities to gur in mouse taste cells, one gur sensitive and the other gur insensitive. Taste cells possessing each component may be specifically innervated by a particular type of chorda tympani neurons. (+info)
Purification of chitinolytic protein from Rehmannia glutinosa showing N-terminal amino acid sequence similarity to thaumatin-like proteins.
We have purified a 21-kDa protein, designated as P1, from Rehmannia glutinosa to homogeneity by ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, and preparative native PAGE. The purified P1 had chitin degradation activity. The N-terminal amino acid sequence of P1 indicated that it is very similar to those of thaumatin and other reported thaumatin-like proteins. (+info)
Selective inhibition of sweetness by the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid.
The purpose of this study was to determine the degree to which the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid (Na-PMP) reduced sweet intensity ratings of 15 sweeteners in mixtures. Na-PMP has been approved for use in confectionary/frostings, soft candy and snack products in the USA at concentrations up to 150 p.p.m. A trained panel evaluated the effect of Na-PMP on the intensity of the following 15 sweeteners: three sugars (fructose, glucose, sucrose), three terpenoid glycosides (monoammonium glycyrrhizinate, rebaudioside-A, stevioside), two dipeptide derivatives (alitame, aspartame), two N-sulfonylamides (acesulfame-K, sodium saccharin), two polyhydric alcohols (mannitol, sorbitol), 1 dihydrochalcone (neohesperidin dihydrochalcone), one protein (thaumatin) and one sulfamate (sodium cyclamate). Sweeteners were tested at concentrations isosweet with 2.5, 5, 7.5 and 10% sucrose in mixtures with two levels of Na-PMP: 250 and 500 p.p.m. In addition, the 15 sweeteners were tested either immediately or 30 s after a pre-rinse with 500 p.p.m. Na-PMP. In mixtures, Na-PMP at both the 250 and 500 p.p.m. levels significantly blocked sweetness intensity for 12 of the 15 sweeteners. However, when Na-PMP was mixed with three of the 15 sweeteners (monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin), there was little reduction in sweetness intensity. Pre-rinsing with Na-PMP both inhibited and enhanced sweetness with the greatest enhancements found for monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin, which were not suppressed by Na-PMP in mixtures. The mixture data suggest that Na-PMP is a selective competitive inhibitor of sweet taste. The finding that pre-treatment can produce enhancement may be due to sensitization of sweetener receptors by Na-PMP. (+info)
Genes expressed in Pinus radiata male cones include homologs to anther-specific and pathogenesis response genes.
We describe the isolation and characterization of 13 cDNA clones that are differentially expressed in male cones of Pinus radiata (D. Don). The transcripts of the 13 genes are expressed at different times between meiosis and microspore mitosis, timing that corresponds to a burst in tapetal activity in the developing anthers. In situ hybridization showed that four of the genes are expressed in the tapetum, while a fifth is expressed in tetrads during a brief developmental window. Six of the seven cDNAs identified in database searches have striking similarity to genes expressed in angiosperm anthers. Seven cDNAs are homologs of defense and pathogen response genes. The cDNAs identified are predicted to encode a chalcone-synthase-like protein, a thaumatin-like protein, a serine hydrolase thought to be a putative regulator of programmed cell death, two lipid-transfer proteins, and two homologs of the anther-specific A9 genes from Brassica napus and Arabidopsis. Overall, our results support the hypothesis that many of the reproductive processes in the angiosperms and gymnosperms were inherited from a common ancestor. (+info)
Intake of umami-tasting solutions by mice: a genetic analysis.
In two-bottle preference tests with water and solutions of monosodium glutamate (MSG) and inosine-5'-monophosphate (IMP), mice from the C57BL/6ByJ inbred strain consumed more and had higher preferences for these solutions compared with mice from the 129/J strain. The C57BL/6ByJ mice consumed 300 mmol/L MSG in large amounts, which were comparable to intakes of highly preferred solutions of sweeteners. The strain differences in voluntary consumption of 300 mmol/L MSG depended at least in part on postingestive effects because prior experience with MSG influenced the expression of the strain difference in MSG acceptance. The strain difference in MSG acceptance was in the opposite direction to the strain difference in NaCl acceptance and was not affected by previous consumption of saccharin. Although the C57BL/6ByJ mice had higher avidity for both MSG and sweeteners than did the 129/J mice, there was no correlation between preferences for these solutions in the second hybrid generation (F(2)) derived from these two strains. Thus, the strain differences in MSG acceptance are not related to the strain differences in salty or sweet taste responsiveness and most likely represent specific umami taste responsiveness. High acceptance of MSG solutions by the C57BL/6ByJ mice was inherited as a recessive trait in the F(2) generation. Further genetic linkage analyses using the F(2) hybrids are being conducted to map chromosomal locations of genes determining the strain difference in MSG acceptance. (+info)
Chewing gum--facts and fiction: a review of gum-chewing and oral health.
The world market for chewing gum is estimated to be 560,000 tons per year, representing approximately US $5 billion. Some 374 billion pieces of chewing gum are sold worldwide every year, representing 187 billion hours of gum-chewing if each piece of gum is chewed for 30 minutes. Chewing gum can thus be expected to have an influence on oral health. The labeling of sugar-substituted chewing gum as "safe for teeth" or "tooth-friendly" has been proven beneficial to the informed consumer. Such claims are allowed for products having been shown in vivo not to depress plaque pH below 5.7, neither during nor for 30 minutes after the consumption. However, various chewing gum manufacturers have recently begun to make distinct health promotion claims, suggesting, e.g., reparative action or substitution for mechanical hygiene. The aim of this critical review--covering the effects of the physical properties of chewing gum and those of different ingredients both of conventional and of functional chewing gum--is to provide a set of guidelines for the interpretation of such claims and to assist oral health care professionals in counseling patients. (+info)