In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expression was also pursued. T2R4 expression on the cell surface was increased 2.5 fold, when its N-terminus was tagged with rhodopsin N-terminal 33 residues (Rho33- T2R4 chimera). In conclusion, work carried out provides novel insights into the mechanisms of T2R activation, and in the discovery of bitter blockers for T2R4.

Thesis (PhD(Agric) (Horticulture))--University of Stellenbosch, 2005. / Bitter pit fruit in commercial consignments of apples still poses an economic threat to exporters from South Africa. Bitter pit develops pre-harvest, but gets progressively worse during storage and is only traceable once the lesions appear after storage. Accurate, early indications of bitter pit incidence will allow for remedial pre-harvest measures in the field, e.g. Ca foliar applications, to reduce the potential losses. Similarly, the automatic detection of a bitter pit fruit during packing will reduce financial losses by identifying unacceptable fruit before shipping. Fluorescence imaging is a fast, non-destructive technique, able to evaluate numerous fruits individually. Results of pre-harvest imaging on apples to identify fruit susceptible to bitter pit showed that pitted fruit were correctly classified, but misclassification of non-pitted fruit with fluorescence imaging was still too high. NIR-spectroscopy point meter readings could distinguish visible bitter pit lesions from healthy tissue. Important wavelengths associated with visible bitter pit were identified. This technique could also identify immature apples, more prone to bitter pit development. It could however not distinguish between bitter pit and non-pitted fruit when applied randomly on the calyx end of apples at harvest. Pre-harvest foliar applications to increase fruit Ca content and reduce bitter pit incidence, is a standard practice world wide. External Ca uptake by fruit was monitored to determine the efficacy of applications during different stages of fruit development. Two periods of efficient uptake of external Ca were identified, viz., cell division and the last few weeks before harvest. Foliar Ca applications from 40 days after full bloom were more effective in increasing fruit Ca content and reducing bitter pit incidence than at 80 days after full bloom, which was recommended previously. Mineral analysis of fruit has been used with variable success to predict bitter pit prior to harvest. The possibility of increasing the accuracy of existing predictive models by using analysis of individual fruit rather than pooled samples, was investigated. By improving the normality of different mineral distributions and decreasing the overlap between pitted and non-pitted fruit classes, it was attempted to improve the reliability of predictions based on variable threshold values. The Ca distribution showed a variation between pitted and nonpitted classes, but still a significant overlap between classes reduced the accuracy of the predictive capacity of this distribution. Even though our results produced a correct classification of 85% for non-pitted fruit, which can be useful, this was still below the required tolerance, of less that 2%, expected on the market. The effect of pruning and fruit bearing position on two-year-old wood on dry mass and Ca allocation of fruit was determined. ‘Golden Delicious’ fruit set was the lowest at the basal bearing position compared to the other positions evaluated and was contrary to expectations. Fruit in a terminal bearing position was superior to the basal position regarding total dry weight and fruit size. Distal wood possibly inhibited growth and set on the basal position via auxin distribution. Ca allocation differed between seasons and cultivars and could either be influenced by bearing position or presence or absence of re-growth.

Magister Scientiae Dentium - MSc(Dent) / Introduction: Dental caries has one the highest incidences in children and the host’s diet may be a major factor in determining susceptibility to the disease. A proposed tool to screen and identify high risk individuals uses a bitter compound 6-n-propylthiouracil (PROP). The goal of this screening tool is to identify mothers and children who are Non-tasters (those who cannot taste PROP) and to educate them about their possible affinity towards sugar substances and its harmful effects on oral and general health. It is suggested that Non-taster children could be prioritized when providing preventative dental treatment. Aim: To validate the use of PROP as a screening tool for determining high caries risk individuals by identifying the taster status of mothers and children, their preference towards sugar, and its impact on their caries status. Methodology: 75 mother/ child pairs were recruited to participate in this study. Caries experience, sugar preference and taster status were determined for all the subjects. Comparisons were made between mothers and their children to find a possible association. Results: Caries experience was greater in individuals who were Non-tasters when compared to Super-tasters. A significant association between taster status and DMFT score was established (p<0.000). A significant association between taster status and sugar preference was established (p<0.000). A positive correlation with regard to taster status, sugar preference and caries experience was observed in mother/child pairs.

Magister Scientiae Dentium - MSc(Dent) / Introduction: Dental caries has one the highest incidences in children and the host’s diet may be a major factor in determining susceptibility to the disease. A proposed tool to screen and identify high risk individuals uses a bitter compound 6-n-propylthiouracil (PROP). The goal of this screening tool is to identify mothers and children who are Non-tasters (those who cannot taste PROP) and to educate them about their possible affinity towards sugar substances and its harmful effects on oral and general health. It is suggested that Non-taster children could be prioritized when providing preventative dental treatment. Aim: To validate the use of PROP as a screening tool for determining high caries risk individuals by identifying the taster status of mothers and children, their preference towards sugar, and its impact on their caries status. Methodology: 75 mother/ child pairs were recruited to participate in this study. Caries experience, sugar preference and taster status were determined for all the subjects. Comparisons were made between mothers and their children to find a possible association. Results: Caries experience was greater in individuals who were Non-tasters when compared to Super-tasters. A significant association between taster status and DMFT score was established (p<0.000). A significant association between taster status and sugar preference was established (p<0.000). A positive correlation with regard to taster status, sugar preference and caries experience was observed in mother/child pairs. Conclusion: Similarities in the mother's and child’s PROP taster status and its association with sugar preference allows such a screening test to identify individuals who are at high risk of developing dental caries. Early identification of mothers who are Non-tasters may allow the introduction of early intervention strategies and assist in the early detection of potentially high-risk children, especially in environments where resources are limited.

T2R receptors are the classical bitter taste receptors which detect and transduce bitter taste in a subset of taste receptor cells (TRCs). The TRPM5-dependent T2Rs are G-protein coupled receptors (GPCRs) and are linked to G protein, gustducin to initiate an intracellular signaling cascade for the transduction of bitter tastants. Nicotine is bitter. However, at present the transduction mechanisms for the detection of nicotine in are poorly understood. Previous studies from our laboratory using TRPM5 knockout (KO) mice demonstrated that the T2R pathway is insufficient in explaining the taste perception of nicotine. TRPM5 KO mice elicited chorda tympani (CT) taste nerve responses to nicotine, albeit significantly smaller than the wild type (WT) mice and still responded to nicotine as an aversive stimulus. Following addition of mecamylamine (Mec), a non-specific blocker of neuronal nicotinic acetylcholine receptors (nAChRs), CT responses to nicotine were partially inhibited in both WT and TRPM5 KO mice. Mec also decreases the aversive response to nicotine in both WT and TRPM5 KO mice. These studies led to the hypothesis that both a TRPM5-independent and TRPM5-dependent pathways are responsible for the detection and transduction of the bitter taste of nicotine in TRCs. The TRPM5-independent pathway most likely utilizes the nAChRs expressed in TRCs and function as bitter taste receptors for nicotine. We have subsequently demonstrated the expression of nAChRs in a subset of TRPM5-positive TRCs. However, this mechanism is not well understood in other cell types, particularly undifferentiated epithelial cells, such as HEK293T cells. The specific aims of this project were: (i) To identify which components of T2R-dependent taste reception as well as components of nAChRs are expressed in HEK293T cells; (ii) To determine if HEK293T cells co-express these components; (iii) To identify if exposure to nicotine modulates the expression of T2R and nAChR dependent components in HEK293T cells; (iv) To determine if TRCs express functional nAChR ion channels; and (v) To determine if nAChRs are involved in the release of neuropeptides, such as brain-derived neurotrophic factor (BDNF) in HEK293T cells. The data obtained in HEK293T cells was compared with parallel studies on adult cultured human fungiform taste cells (HBO) done independently by Dr. Jie Qian, a postdoctoral fellow in Dr. Vijay Lyall’s lab. The results of combined studies on HBO and HEK293T cells indicates that TRPM5-positive cells also co-express ionotropic nAChRs, comprising a and β subunits. The nAChRs are capable of forming ion pores and when stimulated by nicotine and create a parallel TRPM5-independent pathway for the detection of nicotine. Using molecular and immunocytochemical techniques, our results demonstrate that mRNAs and proteins for bitter taste receptors and downstream intracellular signaling components as well as subunits necessary for the formation of nAChRs are expressed in HBO and HEK293T cells. Results demonstrated that TRPM5-positive HEK293T cells co-expressed nAChR subunits throughout the entire population. Nicotine increased the influx of Ca2+ in a dose dependent manner, which was somewhat reduced by the addition of TRPM5 blocker, triphenylphosphine oxide (TPPO). Both mRNA and protein expression were altered in a biphasic pattern with a maximum increased observed at 0.5 µM nicotine with a decrease in expression at higher concentrations. The synthesis of neurotrophic factor BDNF, required for maturation of taste bud cells and their innervating nerves, increased in HEK293T cells exposed to nicotine, however, nicotine did not trigger the release of BDNF. These results were then compared and contrasted with HBO cells to better understand the comparative effects of nicotine on both undifferentiated and differentiated cells. The data on HBO cells is presented in the Appendix.

No description available.

Hubbard Foods Limited of Auckland makes a variety of oats-based value-added products. In the preparation of a range of products at Hubbard Foods, technical staff has become aware of a bitterness problem that sporadically appears in toasted oats. Toasting involves dry heating to about 150°C resulting in the golden colour and flavour development necessary for range of products. Bitterness development has been described in the literature, but Hubbard staff is necessarily focussed on production issues, rather than on a sporadic problem seemingly outside the scope of production variables. The author of this thesis set out to identify the cause and suggest a remedy. Prior research with oats has shown that bitterness and associated off-flavours are linked to the accumulation of free fatty acids, their volatile oxidation products, and possibly amino acids and certain phenols. Oats are distinguished from related grains by their high relative fat content, about seven percent, and an associated very active lipase. The free fatty acids stem from the lipase activity that should be, but may not be, inactivated at source in Australia. This is achieved in the milling process by physical disruption and moist heating to a temperature at which the enzyme is denatured. However, residual lipase activity may adversely affect oats quality during time in storage and transit. A number of analytical methods for cereals were adapted to match the constraints of time and resources. These methods were for colour, moisture, peroxidase activity, p-anisidine, and fat and free fatty acids content, composition of fatty acids, total phenols, volatiles, and bitterness as perceived by an analytical sensory panel of four people. Determination of lipase activity is very expensive, so peroxidase activity is commercially used as an indicator. If the latter is inactive, the former will necessarily be also inactive. The designed methods were first applied to 17 oats lots passing through the Hubbard environment, where 14 were paired raw and toasted. The values of moisture, fat content, free fatty acids content and total phenols were within the normal limits expected for commercial lots of oats compared with the previous studies. Not much variation was observed among the 17 oats lots, with the exception of lot DWHE25. Lot DWHE25 was a faulty product, which had high moisture content, high free fatty acids content, and tasted very bitter. The results suggested that moisture content, free fatty acids and bitterness were usually correlated. In spite of the differences encountered and the clues provided by extremes, the data generated from Hubbard oats lots did not provide enough variation in quality to lead to a definitive chemical model of bitter flavour development. But perhaps crucially, it was found that most samples as received from Hubbard Foods were peroxidase-active which conflicted with the results reported on specification sheets prepared by the oats supplier. These specifications accompanied each lot delivered to Hubbard Foods. Therefore, the supplier’s method was examined and was found to be deficient in one critically important respect. Their method omitted the key reactant hydrogen peroxide. Therefore, it is possible that the lipase was active in many of the samples. Therefore, some experiments were conducted where raw oats, from Hubbard Foods and a supermarket, were treated with water additions and stored for a period to examine the effect of moisture content on the quality and flavour deterioration on subsequent storage. Water-treated oats were toasted to simulate a typical Hubbard process, yielding a total of 58 samples with carrying moisture contents. The data set was statistically analysed to identify the cause of bitterness and the means of its control. The free fatty acids content, volatile compounds particularly hexanal, and total phenols increased with moisture content and storage time. The correlations between chemical analysis and sensory test indicated that free fatty acids positively correlated with bitterness (r = 0.71), and hexanal was also positively correlated. Total phenols did not appear to correlate with bitterness. Oats lots with high peroxidase activity tended to have the poorest quality, strongly implicating residual lipase activity as the critical factor. There were no important interactions between water addition and toasting for most of the experiments. Therefore, it seems likely that the toasting procedures at Hubbard Foods are not responsible for bitterness formation. The cause(s) of bitterness is certainly at source, with a faulty peroxidase test strongly implicated.

Hubbard Foods Limited of Auckland makes a variety of oats-based value-added products. In the preparation of a range of products at Hubbard Foods, technical staff has become aware of a bitterness problem that sporadically appears in toasted oats. Toasting involves dry heating to about 150°C resulting in the golden colour and flavour development necessary for range of products. Bitterness development has been described in the literature, but Hubbard staff is necessarily focussed on production issues, rather than on a sporadic problem seemingly outside the scope of production variables. The author of this thesis set out to identify the cause and suggest a remedy. Prior research with oats has shown that bitterness and associated off-flavours are linked to the accumulation of free fatty acids, their volatile oxidation products, and possibly amino acids and certain phenols. Oats are distinguished from related grains by their high relative fat content, about seven percent, and an associated very active lipase. The free fatty acids stem from the lipase activity that should be, but may not be, inactivated at source in Australia. This is achieved in the milling process by physical disruption and moist heating to a temperature at which the enzyme is denatured. However, residual lipase activity may adversely affect oats quality during time in storage and transit. A number of analytical methods for cereals were adapted to match the constraints of time and resources. These methods were for colour, moisture, peroxidase activity, p-anisidine, and fat and free fatty acids content, composition of fatty acids, total phenols, volatiles, and bitterness as perceived by an analytical sensory panel of four people. Determination of lipase activity is very expensive, so peroxidase activity is commercially used as an indicator. If the latter is inactive, the former will necessarily be also inactive. The designed methods were first applied to 17 oats lots passing through the Hubbard environment, where 14 were paired raw and toasted. The values of moisture, fat content, free fatty acids content and total phenols were within the normal limits expected for commercial lots of oats compared with the previous studies. Not much variation was observed among the 17 oats lots, with the exception of lot DWHE25. Lot DWHE25 was a faulty product, which had high moisture content, high free fatty acids content, and tasted very bitter. The results suggested that moisture content, free fatty acids and bitterness were usually correlated. In spite of the differences encountered and the clues provided by extremes, the data generated from Hubbard oats lots did not provide enough variation in quality to lead to a definitive chemical model of bitter flavour development. But perhaps crucially, it was found that most samples as received from Hubbard Foods were peroxidase-active which conflicted with the results reported on specification sheets prepared by the oats supplier. These specifications accompanied each lot delivered to Hubbard Foods. Therefore, the supplier’s method was examined and was found to be deficient in one critically important respect. Their method omitted the key reactant hydrogen peroxide. Therefore, it is possible that the lipase was active in many of the samples. Therefore, some experiments were conducted where raw oats, from Hubbard Foods and a supermarket, were treated with water additions and stored for a period to examine the effect of moisture content on the quality and flavour deterioration on subsequent storage. Water-treated oats were toasted to simulate a typical Hubbard process, yielding a total of 58 samples with carrying moisture contents. The data set was statistically analysed to identify the cause of bitterness and the means of its control. The free fatty acids content, volatile compounds particularly hexanal, and total phenols increased with moisture content and storage time. The correlations between chemical analysis and sensory test indicated that free fatty acids positively correlated with bitterness (r = 0.71), and hexanal was also positively correlated. Total phenols did not appear to correlate with bitterness. Oats lots with high peroxidase activity tended to have the poorest quality, strongly implicating residual lipase activity as the critical factor. There were no important interactions between water addition and toasting for most of the experiments. Therefore, it seems likely that the toasting procedures at Hubbard Foods are not responsible for bitterness formation. The cause(s) of bitterness is certainly at source, with a faulty peroxidase test strongly implicated.

Se estudia la nutrición mineral en manzanas libres y afectadas por bitter pit, de las variedades: Fuji Royal, Granny Smith y Fuji. Los elementos esenciales determinados, en cáscara y pulpa, son: Cu, Zn, Fe, Mn, Ca, Mg, K y P. También se incluye Sr, elemento no esencial, del cual se dispone poca información sobre este tema. Inicialmente los frutos fueron evaluados determinando: peso del fruto, firmeza de la pulpa, concentración de sólidos solubles y acidez titulable. El análisis de los datos se centró especialmente en las diferencias entre frutos libres y afectados por bitter pit. Se detecta una menor concentración de calcio en frutos afectados en comparación con los libres del desorden fisiológico, confirmando lo concluido por otros autores. Sin embargo, respecto al magnesio solo la variedad Fuji presenta una relación con el daño. También se detecta y confirma lo obtenido anteriormente para la variedad Fuji, donde unido a la deficiencia de calcio se presenta una mayor concentración de potasio en frutos afectados por el daño, lo cual no fue observado en las otras variedades. El estroncio, se encuentra en frutos en un rango de concentraciones de 1 a 4 µg/gps. Sin embargo, el contenido de estroncio es significativamente menor en cáscara de frutos afectados que en los libres tanto en variedad Fuji Royal como en Granny Smith. De acuerdo a los datos obtenidos, y en relación a la nutrición mineral equilibrada de las plantas, las relaciones binarias y terciarias resultan ser mejores indicadores para incidencia de bitter pit que la concentración de cada mineral en forma independiente. Estas relaciones son: Mg/Ca, K/Ca y (Mg + K)/ Ca. Finalmente, es necesario destacar que la cáscara es el tejido más adecuado para estudiar la nutrición mineral en manzana respecto al bitter pit.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13104号 / 論農博第2850号 / 新制||農||1051(附属図書館) / 学位論文||H29||N5036(農学部図書室) / (主査)教授 森 直樹, 教授 宮川 恒, 教授 三芳 秀人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM