Nutrients Amino Acids Research Articles

Available versus digestible amino acids – new stable isotope methods

The nutritive value of food protein sources is dependent on the amino acid composition and the bioavailability of the nutritionally indispensable amino acids. Traditionally the methods developed to determine amino acid bioavailability have focused on intestinal absorption or digestibility, which is calculated as the percent of amino acid intake that does not appear in digesta or faeces. Traditional digestibility based methods do not always account for gut endogenous amino acid losses or absorbed amino acids which are unavailable due to the effect of heat processing and the presence of anti-nutritional factors, though methods have been developed to address these issues. Furthermore, digestibility based methods require the use of animal models, thus there is a need to develop in vivo methods that can be applied directly in human subjects to identify the proportion of dietary amino acids which is bioavailable, or metabolically available to the body for protein synthesis following digestion and absorption. The indicator amino acid oxidation (IAAO) method developed in our laboratory for humans has been systematically applied to determine almost all indispensable amino acid requirements in adult humans. Oxidation of the indicator amino acid is inversely proportional to whole body protein synthesis and responds rapidly to changes in the bioavailability of amino acids for metabolic processes. Using the IAAO concept, we developed a new in vivo method in growing pigs, pregnant sows and adult humans to identify the metabolic availability of amino acids in foods. The stable isotope based metabolic availability method is suitable for rapid and routine analysis in humans, and can be used to integrate amino acid requirement data with dietary amino acid availability of foods.

Gut luminal endogenous protein: Implications for the determination of ileal amino acid digestibility in humans

The true ileal digestibility assay provides the most informative measure of digestibility to assess bioavailability of amino acids in foods for humans. To determine 'true' estimates of ileal amino acid digestibility, requires that endogenous amino acids present in digesta at the terminal ileum be quantified. The amounts of endogenous amino acids in ileal digesta can be determined after feeding an animal or human a protein-free diet (traditional approach) or by various methods after giving a protein-containing diet. When the protein-free method has been applied with adult human subjects an overall mean value (three separate studies) for endogenous ileal nitrogen flow of 800 mg N/d has been reported. This value is considerably lower than a comparable value obtained after feeding protein of 1852 mg N/d (mean of four separate studies), and thus endogenous ileal N and amino acids should be measured under conditions of protein alimentation. There is some confusion concerning the terminology used to define digestibility, with the term "true" digestibility having different adopted meanings. Here, true amino acid digestibility is defined as apparent amino acid digestibility corrected for the basal amino acid losses determined after giving either a protein-free or a protein-containing diet. Basal losses should be determined at a defined dry-matter and protein intake. The protein-free diet approach to determining endogenous amino acids is considered unphysiological and basal losses refer to ileal endogenous amino acid flows associated with digesta dry-matter flow, and not including "specific" effects of dietary factors such as non starch polysaccharides and anti nutritional factors. Arguments are advanced that the enzyme hydrolysed protein/ultra filtration method may be suitable for routine application with a cannulated pig model, to obtain physiologically-valid basal estimates of ileal endogenous amino acids to allow calculation of true ileal amino acid digestibility in the pig, and then prediction (via statistical relationships) of true coefficients of amino acid digestibility in humans.

Amino acid sensing mechanisms: an Achilles heel in cancer?

The act of increasing mass, either in non-dividing cells or in dividing cells seeking to provide new material for daughter cells, depends upon the continued presence of extracellular nutrients in order to conserve mass. For amino acid nutrients, it appears that their insufficiency for new protein synthesis is actively monitored by both prokaryotic and eukaryotic cells, eliciting appropriate cellular responses that may depend not only on bulk nutrient supply, but also on the abundance of specific amino acids.

English

Biogenic amines are formed in some foods, especially fish and fish products as a result of the process of decarboxylation of free amino acids in food. The histamine (scombroid) poisoning arises from ingestion foods containing relatively high levels of histamine and other amines that act as histamine potentiators. Therefore, this study aims to explore the level of contamination by biogenic amines in fish and fish products in shore of Thuel – Saudi Arabia. One hundred and twenty of different fish samples were collected from Thuel shore markets. The biogenic amines histamine, cadaverine, putrescine and tyramine were determined in all samples and also in selected fish samples held iced for 0, 24 and 48 h. The effect of the commercial frying process on biogenic amines level was also tested using two different types of fish. Quantitative thin layer chromatography (TLC) analysis was used in biogenic amines determination. There are significant differences between the tested samples and the highest mean (mg/100 g) of putrescine. Cadaverine, histamine and tyramine were in loat fish (6.57), Baiad (3.348), Hareed (4.82) and Baiad (1.38), respectively. Significant increases in the level of biogenic amines were observed in fish held iced at 0, 24, and 48 h. A significant increase of histamine level was noticed in selected fish after commercial frying process. Key words: Biogenic amines, fish, frying, hazard analysis critical control point (HACCP),thin layer chromatography (TLC).

Rapid determination of amino acids in foods by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry

This study describes a rapid and sensitive analytical method for the determination of amino acids in foods and drinks. The method entailed dilution or extraction of amino acids from foods using the mixture of acetonitrile and 0.1% aqueous formic acid (50:50, v/v). Chromatographic separation of underivatized amino acids was performed using a hydrophilic interaction liquid chromatography within a runtime of 6 min. Both hydrophobicity and charge of the side chain played important roles on the elution order of amino acids under the chromatographic conditions. High-resolution mass spectrometry allowed qualitative and quantitative detection of amino acids in complex food matrices. Its response was found linear over a concentration range of 0.25-10 μg/ml. The method could be successfully applied to various foods and drinks to profile individual amino acids. Mean percentage recoveries of amino acids from different matrices were 88.5% or higher with residual standard deviation of less than 5.0%.

A Novel Biorational Pesticide: Efficacy of Methionine Against Heraclides (Papilio) cresphontes, a Surrogate of the Invasive Princeps (Papilio) demoleus (Lepidoptera: Papilionidae)

ABSTRACT The Southeast Asian citrus-feeding butterfly Princeps (Papilio) demoleus (L.) was recently introduced into the Americas, causing an imminent threat to citrus production and ornamental flora. The human nutrient amino acid methionine has been shown by us to disrupt aminoacid-modulated ion transport systems in caterpillars and other insect larvae that possess an alkaline midgut. Heraclides (Papilio) cresphontes was bioassayed as a United States Department of Agriculture permitted surrogate of the Florida quarantined P. demoleus to test the potential efficacy of methionine. Larvae were allowed to feed ad libitum on wild lime plants with leaves treated with methionine or proline. Methionine caused 100% mortality in first through fourth instars in a time- and dose-dependent manner, as determined by probit analysis whereas proline was not toxic. Wild lime host plants did not exhibit phytotoxicity with methionine treatments during a 14 d test period. It is concluded that methionine is an effective larvicide against H. cresphontes, and therefore may be a candidate environmentally safe biorational pesticide for use against invasive P. demoleus in the Americas.

Comparison on Nutrient Composition and Amino Acid and Fatty Acid Profile of Muscle in Wild and Farmed Prawn, <i>Penaeus japonicu</i>

Penaeus japonicus is an important marine shrimps resource in China. In order to evaluate the nutritional value, the contents of general nutritional compositions, amino acids and fatty acids in muscle were compared between wild and farmed P. japonicus. The contents of muscle moisture and crude protein, fat and ash in wild P. japonicus were 77.16%, 17.74%, 2.08% and 1.79％, and those in farmed P. japonicus were 78.02%, 17.26%, 2.04% and 1.63％, respectively. The essential amino acids (EAA) in wild and farmed P. japonicus were 23.25％ and 22.43％, respectively. The amino acid score of essential amino acids were higher than 100 except Leu and Trp, so the protein of P. japonicus has a well-balanced amino acid composition. Wild P. japonicus has more unsaturated fatty acids (64.05％) than farmed group (60.34％). Higher n-3 polyunsaturated fatty acids (PUFAs), eicosapentainoic acids (EPA, 20:5 n-3), docosahexaenoic acids (DHA, 22:6 n-3) contents and lower C16:0, C20:0, C18:1, C18:2, C18:3 levels were detected in the wild group. Values of n-6/n-3 in muscle of farmed and wild P. japonicus were 0.30 and 0.23 respectively, which were both significantly lower than the maximum value (4.0) recommended by UK Department of Health (HMSO, 1994), while Values of the PUFA/SFA ratio of two groups were 0.60 and 0.74 higher than the minimum value (0.45) recommended by HMSO. Thus the muscle of farmed and wild P. japonicus are both healthy and safe, and the the muscle of wild P. japonicus is more beneficial to human health.

Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion

BackgroundA major goal of molecular evolutionary biology is to understand the fate and consequences of duplicated genes. In this context, aphids are intriguing because the newly sequenced pea aphid genome harbors an extraordinary number of lineage-specific gene duplications relative to other insect genomes. Though many of their duplicated genes may be involved in their complex life cycle, duplications in nutrient amino acid transporters appear to be associated rather with their essential amino acid poor diet and the intracellular symbiosis aphids rely on to compensate for dietary deficits. Past work has shown that some duplicated amino acid transporters are highly expressed in the specialized cells housing the symbionts, including a paralog of an aphid-specific expansion homologous to the Drosophila gene slimfast. Previous data provide evidence that these bacteriocyte-expressed transporters mediate amino acid exchange between aphids and their symbionts.ResultsWe report that some nutrient amino acid transporters show male-biased expression. Male-biased expression characterizes three paralogs in the aphid-specific slimfast expansion, and the male-biased expression is conserved across two aphid species for at least two paralogs. One of the male-biased paralogs has additionally experienced an accelerated rate of non-synonymous substitutions.ConclusionsThis is the first study to document male-biased slimfast expression. Our data suggest that the male-biased aphid slimfast paralogs diverged from their ancestral function to fill a functional role in males. Furthermore, our results provide evidence that members of the slimfast expansion are maintained in the aphid genome not only for the previously hypothesized role in mediating amino acid exchange between the symbiotic partners, but also for sex-specific roles.

Cover Picture: Mol. Nutr. Food Res. 9'2011

AbstractThis special issue of Molecular Nutrition & Food Research is dedicated to acrolein (2‐propenal), a constituent of many foods that has poorly understood long‐term effects on human health. Acrolein is primarily formed from carbohydrates, lipids and certain amino acids in foods by heat treatment. Besides intake of acrolein via the diet, humans are exposed to acrolein through inhalation of vapors emitted from heated cooking oils, cigarette smoke, and inhalation of combustion products of fossil fuels. Finally, acrolein is formed endogenously as a product of polyamine metabolism and oxidative stressThese and other aspects are covered in this special issue by nine reviews and three research articles.

Acrolein

This special issue of Molecular Nutrition and Food Research is dedicated to acrolein (2-propenal), a constituent of many foods that has poorly understood long-term effects on human health. It is therefore not a surprise that the number of publications on acrolein has steadily increased from 61 publications per year in 2000 to 197 in 2010 (source: http://gopubmed.org/). Acrolein is primarily formed from carbohydrates, lipids and certain amino acids in foods by heat treatment. Besides intake of acrolein via the diet, humans are exposed to acrolein through inhalation of vapors emitted from heated cooking oils, cigarette smoke and inhalation of combustion products of fossil fuels. Finally, acrolein is formed endogenously as a product of polyamine metabolism and oxidative stress. The adverse effects of acrolein on human health originate from its capability to form Michael-type adducts with cysteine, lysine and histidine residues in proteins and with amine groups in DNA bases. These and other aspects are covered in this special issue by nine reviews and three research articles. Abraham et al. (this issue) provide an overview of the metabolism of acrolein, its toxicity and carcinogenicity and its occurrence in various foods and beverages. They conclude that exposure to acrolein via the diet probably does not pose immediate health risks, but, at the same time, point out that a risk assessment is difficult to make due to technical difficulties with measuring acrolein levels in foods. An accurate risk assessment is further complicated by lack of knowledge regarding the immediate and especially the long-term consequences of acrolein's ability to interfere with many metabolic pathways and (redox-sensitive) signaling networks. The contributions by Bein and Leikauf, Conklin et al., Fabisiak et al. and by Myers et al. provide a wealth of new information with respect to the effects of acrolein on gene regulation. Other contributions emphasize the chemical diversity of the cellular damage exerted by acrolein and the molecular consequences of acrolein exposure in cultured cells, laboratory animals and humans. Knowing how and to what extent acrolein modifies DNA and proteins is necessary for elucidating the mechanisms by which acrolein causes cellular dysfunction. The mechanisms of acrolein-related toxicity that affect the nervous system (spinal cord trauma), the brain and the lung are discussed in this issue by experts in the field. Also discussed are strategies aimed at mitigating the deleterious effects of acrolein by using acrolein scavengers such as flavonoids and other (dietary) phytochemicals, vitamins, neutraceuticals and pharmaceuticals. Progress in understanding acrolein's impact on health would not have been possible without the advent of new analytical technologies. Recent analytical breakthroughs include the development of acrolein-specific immunochemical techniques and microarray technologies for assessing acrolein toxicity. Substantial progress has been achieved in assessing acrolein exposure at the molecular level in vivo. Modern mass spectrometry-based techniques enable the analysis of acrolein-mediated DNA damage (reviewed by Chen in this issue) and the analysis of acrolein adducts in mitochondrial proteomes (Wu et al.). This special issue on acrolein offers an overview of the astounding progress that has been made in unraveling the multifaceted effects of acrolein on biological systems and human health. Despite the advances made in understanding the biological consequences of acute acrolein exposure, many questions remain unanswered regarding the health impact of chronic acrolein exposure via the diet and the environment. We agree with the contributors of this issue that further progress in this area requires the development of methods for reliable quantification of acrolein in foods and beverages as well as methods for probing the interaction of acrolein with its biological environment. We are grateful to the Editor-in-Chief for providing us the opportunity to invite distinguished authors to contribute to this issue, to the reviewers for their invaluable comments and to the Editorial Office for tireless support in smoothing the path towards completion of this special issue. Jan Frederik Stevens Oregon State University Claudia S. Maier Oregon State University

Insulin-like peptide genes in honey bee fat body respond differently to manipulation of social behavioral physiology.

Nutrient sensitive insulin-like peptides (ILPs) have profound effects on invertebrate metabolism, nutrient storage, fertility and aging. Many insects transcribe ILPs in specialized neurosecretory cells at changing levels correlated with life history. However, the major site of insect metabolism and nutrient storage is not the brain, but rather the fat body, where functions of ILP expression are rarely studied and poorly understood. Fat body is analogous to mammalian liver and adipose tissue, with nutrient stores that often correlate with behavior. We used the honey bee (Apis mellifera), an insect with complex behavior, to test whether ILP genes in fat body respond to experimentally induced changes of behavioral physiology. Honey bee fat body influences endocrine state and behavior by secreting the yolk protein precursor vitellogenin (Vg), which suppresses lipophilic juvenile hormone and social foraging behavior. In a two-factorial experiment, we used RNA interference (RNAi)-mediated vg gene knockdown and amino acid nutrient enrichment of hemolymph (blood) to perturb this regulatory module. We document factor-specific changes in fat body ilp1 and ilp2 mRNA, the bee's ILP-encoding genes, and confirm that our protocol affects social behavior. We show that ilp1 and ilp2 are regulated independently and differently and diverge in their specific expression-localization between fat body oenocyte and trophocyte cells. Insect ilp functions may be better understood by broadening research to account for expression in fat body and not only brain.

Signalling by amino acid nutrients

It is clear that mTORC1 (mammalian target of rapamycin complex 1) is regulated by the presence of ambient amino acid nutrients. However, the mechanism by which amino acids regulate mTORC1 is still open to question, despite extensive efforts. Our recent work has revealed that PR61ϵ, a B56 family regulatory subunit of PP2A (protein phosphatase 2A), associates with and regulates the activity of MAP4K3 (mitogen-activated protein kinase kinase kinase kinase 3), a protein kinase regulated by amino acid sufficiency that acts upstream of mTORC1. In searching for a physiological process regulated by amino acids, we have demonstrated recently that arginine plays a role in the activation of LPS (lipopolysaccharide)-induced MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling in macrophages. PP2A similarly associates with the upstream regulator of MEK in this signalling pathway, TPL-2 (tumour progression locus-2), in response to arginine availability. Thus PP2A is a negative regulator of both MAP4K3 and TPL-2 in both mTORC1 and MEK/ERK signalling pathways.

Amino Acid Signaling to mTOR Mediated by Inositol Polyphosphate Multikinase

mTOR complex 1 (mTORC1; mammalian target of rapamycin [mTOR] in complex with raptor) is a key regulator of protein synthesis and cell growth in response to nutrient amino acids. Here we report that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid kinase activities, regulates amino acid signaling to mTORC1. This regulation is independent of IPMK's catalytic function, instead reflecting its binding with mTOR and raptor, which maintains the mTOR-raptor association. Thus, IPMK appears to be a physiologic mTOR cofactor, serving as a determinant of mTORC1 stability and amino acid-induced mTOR signaling. Substances that block IPMK-mTORC1 binding may afford therapeutic benefit in nutrient amino acid-regulated conditions such as obesity and diabetes.

Prenatal testosterone-induced fetal growth restriction is associated with down-regulation of rat placental amino acid transport

BackgroundExposure of pregnant mothers to elevated concentrations of circulating testosterone levels is associated with fetal growth restriction and delivery of small-for-gestational-age babies. We examined whether maternal testosterone crosses the placenta to directly suppress fetal growth or if it modifies placental function to reduce the capacity for transport of nutrients to the fetus.MethodsPregnant rats were exposed to testosterone propionate (TP; 0.5 mg/kg) by daily subcutaneous injection from gestational days (GD) 15-19. Maternal and fetal testosterone levels, placental nutrient transport activity and expression of transporters and birth weight of pups and their anogenital distances were determined.ResultsThis dose of TP doubled maternal testosterone levels but had no effect on fetal testosterone levels. Maternal daily weight gain was significantly lower only on GD 19 in TP treated dams compared to controls. Placental weight and birth weight of pups were significantly reduced, but the anogenital distance of pups were unaffected by TP treatment. Maternal plasma amino acids concentrations were altered following testosterone exposure, with decreases in glutamine, glycine, tyrosine, serine, proline, and hydroxyproline and increases in asparagine, isoleucine, leucine, lysine, histidine and arginine. In the TP dams, placental system A amino acid transport activity was significantly reduced while placental glucose transport capacity was unaffected. Decreased expression of mRNA and protein levels of slc38a2/Snat2, an amino acid transporter, suggests that reduced transporter proteins may be responsible for the decrease in amino acid transport activity.ConclusionsTaken together, these data suggest that increased maternal testosterone concentrations do not cross the placenta to directly suppress fetal growth but affects amino acid nutrient delivery to the fetus by downregulating specific amino acid transporter activity.

A Matter Of Degrees: Advancing Our Understanding of Acrylamide

Until about a decade ago, acrylamide was known only as a constituent of cigarette smoke and of products such as plastics and water treatment chemicals. But in 2002 Swedish scientists were surprised to find this human neurotoxicant and probable carcinogen in many heat-processed foods, especially starchy ones such as potato chips, crackers, and french fries. A flurry of research since then has yielded general advice about reducing formation of acrylamide and other heat-generated food toxicants in home cooking and a few recommendations for healthier eating. Now, in 2010, new acrylamide studies are giving a clearer picture of the extent of exposure to the chemical in the United States. These studies also raise additional questions about whether differences in metabolism make exposure more dangerous in certain populations, including children and people who are obese.

Modelling egg production and nutrient responses in broiler breeder hens

SUMMARYThe model described simulates the response of a population of broiler breeders to a daily allowance of a feed of specified composition for up to 280 days from sexual maturity. The population is generated using normally distributed values for initial bodyweight, initial body lipid and protein weight, age at first egg, yolk weight constant and ‘aggressiveness’, the latter producing different feed intakes around the controlled mean. Age at sexual maturity for each bird is predicted from bodyweight and from information about rearing lighting programmes. Egg production, clutch patterns, yolk weights, egg component weights, double-yolked eggs, internal laying and soft-shelled eggs are predicted. Body protein growth, including feathers, is assumed to cease at sexual maturity, thus bodyweight changes after maturity are due entirely to accumulation or utilization of lipid.Energy transactions are expressed in units of effective energy. Protein required for egg production is calculated from the composition of the next egg to be laid and an assumed fixed chemical composition. Assumptions for protein partition rules are that maintenance has highest priority, yolk protein deposition second and albumen protein third. Provided that sufficient energy and nutrients are available, yolk protein deposition is assumed to occur continuously to meet the predicted growth rate, unless inter-clutch intervals exceed 2 days (user defined), when yolk growth ceases. Energy and nutrients (amino acids) for albumen production accumulate in a pool which has some controlling influence over ovulation. If the energy and nutrients in the albumen pool will support the predicted development of the next egg then ovulation will occur, otherwise ovulation is delayed until sufficient energy and nutrients are available. All events are timed within a 24 h day assuming that feeding is at one time in the morning.The desired feed intake is calculated each day according to the supply of the first limiting energy or nutrient (invariably an amino acid) in relation to potential need. Actual feed intake is either the desired intake or the feed allowance, whichever is the lower. The additional energy required for thermogenesis is taken into account, but not the constraining effects of high temperature or the bulkiness of the food as a constraint to voluntary food intake.Some shortcomings of the current model are discussed, these being areas suitable for further research on broiler breeder nutrition.

Evaluation of a novel food composition database that includes glutamine and other amino acids derived from gene sequencing data

To determine the content of glutamine in major food proteins. We used a validated 131-food item food frequency questionnaire (FFQ) to identify the foods that contributed the most to protein intake among 70,356 women in the Nurses' Health Study (NHS, 1984). The content of glutamine and other amino acids in foods was calculated based on protein fractions generated from gene sequencing methods (Swiss Institute of Bioinformatics) and compared with data from conventional (USDA) and modified biochemical (Khun) methods. Pearson correlation coefficients were used to compare the participants' dietary intakes of amino acids by sequencing and USDA methods. The glutamine content varied from 0.01 to to 9.49 g/100 g of food and contributed from 1 to to 33% of total protein for all FFQ foods with protein. When comparing the sequencing and Kuhn's methods, the proportion of glutamine in meat was 4.8 vs 4.4%. Among NHS participants, mean glutamine intake was 6.84 (s.d.=2.19) g/day and correlation coefficients for amino acid between intakes assessed by sequencing and USDA methods ranged from 0.94 to 0.99 for absolute intake, -0.08 to 0.90 after adjusting for 100 g of protein, and 0.88 to 0.99 after adjusting for 1000 kcal. The between-person coefficient of variation of energy-adjusted intake of glutamine was 16%. These data suggest that (1) glutamine content can be estimated from gene sequencing methods and (2) there is a reasonably wide variation in energy-adjusted glutamine intake, allowing for exploration of glutamine consumption and disease.

A putative amino acid transporter of the solute carrier 6 family is upregulated by lithium and is required for resistance to lithium toxicity in Drosophila

Lithium is an efficacious drug for the treatment of mood disorders, and its application is also considered a potential therapy for brain damage. However, the mechanisms underlying lithium's therapeutic action and toxic effects in the nervous system remain largely elusive. Here we report on the use of a versatile genetic model, the fruit fly Drosophila melanogaster, to discover novel molecular components involved in the lithium-responsive neurobiological process. We previously identified CG15088, which encodes a putative nutrient amino acid transporter of the solute carrier 6 (SLC6) family, as one of the genes most significantly upregulated in response to lithium treatment. This gene was the only SLC6 gene induced by lithium, and was thus designated as Lithium-inducible SLC6 transporter or List. Either RNA interference (RNAi)–mediated knockdown or complete deletion of List resulted in a remarkable increase in the susceptibility of adult flies to lithium's toxic effects, whereas transgenic expression of wild-type List significantly suppressed the lithium hypersensitive phenotype of List-deficient flies. Other ions such as sodium, potassium and chloride did not induce List upregulation, nor did they affect the viability of flies with suppressed List expression. These results indicate that lithium's biochemical or physical properties, rather than general osmotic responses, are responsible for the lithium-induced upregulation of List, as well as for the lithium-susceptible phenotype observed in List knockdown flies. Interestingly, flies became significantly more susceptible to lithium toxicity when List RNAi was specifically expressed in glia than when it was expressed in neurons or muscles, which is consistent with potential glial expression of List. These results show that the List transporter confers resistance to lithium toxicity, possibly as a consequence of its amino acid transporter activity in CNS glia. Our results have provided a new avenue of investigation toward a better understanding of the molecular and cellular mechanisms that underlie lithium-responsive neurobiological process.

Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters.

This review provides alternatives to two well established theories regarding membrane energization by H(+) V-ATPases. Firstly, we offer an alternative to the notion that the H(+) V-ATPase establishes a protonmotive force (pmf) across the membrane into which it is inserted. The term pmf, which was introduced by Peter Mitchell in 1961 in his chemiosmotic hypothesis for the synthesis of ATP by H(+) F-ATP synthases, has two parts, the electrical potential difference across the phosphorylating membrane, Deltapsi, and the pH difference between the bulk solutions on either side of the membrane, DeltapH. The DeltapH term implies three phases - a bulk fluid phase on the H(+) input side, the membrane phase and a bulk fluid phase on the H(+) output side. The Mitchell theory was applied to H(+) V-ATPases largely by analogy with H(+) F-ATP synthases operating in reverse as H(+) F-ATPases. We suggest an alternative, voltage coupling model. Our model for V-ATPases is based on Douglas B. Kell's 1979 'electrodic view' of ATP synthases in which two phases are added to the Mitchell model - an unstirred layer on the input side and another one on the output side of the membrane. In addition, we replace the notion that H(+) V-ATPases normally acidify the output bulk solution with the hypothesis, which we introduced in 1992, that the primary action of a H(+) V-ATPase is to charge the membrane capacitance and impose a Deltapsi across the membrane; the translocated hydrogen ions (H(+)s) are retained at the outer fluid-membrane interface by electrostatic attraction to the anions that were left behind. All subsequent events, including establishing pH differences in the outside bulk solution, are secondary. Using the surface of an electrode as a model, Kell's 'electrodic view' has five phases - the outer bulk fluid phase, an outer fluid-membrane interface, the membrane phase, an inner fluid-membrane interface and the inner bulk fluid phase. Light flash, H(+) releasing and binding experiments and other evidence provide convincing support for Kell's electrodic view yet Mitchell's chemiosmotic theory is the one that is accepted by most bioenergetics experts today. First we discuss the interaction between H(+) V-ATPase and the K(+)/2H(+) antiporter that forms the caterpillar K(+) pump, and use the Kell electrodic view to explain how the H(+)s at the outer fluid-membrane interface can drive two H(+) from lumen to cell and one K(+) from cell to lumen via the antiporter even though the pH in the bulk fluid of the lumen is highly alkaline. Exchange of outer bulk fluid K(+) (or Na(+)) with outer interface H(+) in conjunction with (K(+) or Na(+))/2H(+) antiport, transforms the hydrogen ion electrochemical potential difference, mu(H), to a K(+) electrochemical potential difference, mu(K) or a Na(+) electrochemical potential difference, mu(Na). The mu(K) or mu(Na) drives K(+)- or Na(+)-coupled nutrient amino acid transporters (NATs), such as KAAT1 (K(+) amino acid transporter 1), which moves Na(+) and an amino acid into the cell with no H(+)s involved. Examples in which the voltage coupling model is used to interpret ion and amino acid transport in caterpillar and larval mosquito midgut are discussed.

Cloning and functional expression of the first eukaryotic Na+–tryptophan symporter, AgNAT6

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K(0.5)(Trp)=1.3 micromol l(-1) vs K(0.5)(Phe)=430 micromol l(-1)) and [2 or 1 Na(+) or K(+)]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.