A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

PTHR1 polymorphisms influence BMD variation through effects on the growing skeleton

We investigated whether polymorphisms in PTHR1 are associated with bone mineral density (BMD), to determine whether the association of this gene with BMD was due to effects on attainment of peak bone mass or effects on subsequent bone loss. The PTHR1 gene, including its 14 exons, their exon-intron boundaries, and 1,500 bp of its promoter region, was screened for polymorphisms by denaturing high-performance liquid chromatography (dHPLC) and sequencing in 36 osteoporotic cases. Eleven single-nucleotide polymorphisms (SNPs), one tetranucleotide repeat, and one tetranucleotide deletion were identified. A cohort of 634 families, including 1,236 men (39%) and 1,926 women (61%) ascertained with probands with low BMD (Z< -2.0) and the Children in Focus subset of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (785 unrelated individuals, mean age 118 months), were genotyped for the five most informative SNPs (minor allele frequency >5%) and the tetranucleotide repeat. In our osteoporosis families, association was noted between lumbar spine BMD and alleles of a known functional tetranucleotide repeat (U4) in the PTHR1 promoter region (P = 0.042) and between two and three marker haplotypes of PTHR1 polymorphisms with lumbar spine, femoral neck, and total hip BMD (P = 0.021-0.047). This association was restricted to the youngest tertile of the population (age 16-39 years, P = 0.013-0.048). A similar association was found for the ALSPAC cohort: two marker haplotypes of SNPs A48609T and C52813T were associated with height (P = 0.006) and total body less head BMD (P = 0.02), corrected for age and gender, confirming the family findings. These findings suggest a role for PTHR1 variation in determining peak BMD.

Combining HPLC-DAD and ICP-MS data for improved analysis of complex samples: Classification of the root samples from Cortex moutan

A combined data matrix consisting of high performance liquid chromatography–diode array detector (HPLC–DAD) and inductively coupled plasma-mass spectrometry (ICP-MS) measurements of samples from the plant roots of the Cortex moutan (CM), produced much better classification and prediction results in comparison with those obtained from either of the individual data sets. The HPLC peaks (organic components) of the CM samples, and the ICP-MS measurements (trace metal elements) were investigated with the use of principal component analysis (PCA) and the linear discriminant analysis (LDA) methods of data analysis; essentially, qualitative results suggested that discrimination of the CM samples from three different provinces was possible with the combined matrix producing best results. Another three methods, K-nearest neighbor (KNN), back-propagation artificial neural network (BP-ANN) and least squares support vector machines (LS-SVM) were applied for the classification and prediction of the samples. Again, the combined data matrix analyzed by the KNN method produced best results (100% correct; prediction set data). Additionally, multiple linear regression (MLR) was utilized to explore any relationship between the organic constituents and the metal elements of the CM samples; the extracted linear regression equations showed that the essential metals as well as some metallic pollutants were related to the organic compounds on the basis of their concentrations

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Spatial and temporal distribution of tropical phytoplankton species and biomass in the Gulf of Carpenteria, Australia

The biomass and species composition of tropical phytoplankton in Albatross Bay, Gulf of Carpentaria, northern Australia, were examined monthly for 6 yr (1986 to 1992). Chlorophyll a (chl a) concentrations were highest (2 to 5.7 mu g l(-1)) in the wet season at inshore sites, usually coinciding with low salinities (30 to 33 ppt) and high temperatures (29 to 32 degrees C). At the offshore sites chi a concentrations were lower (0.2 to 2 mu g l(-1)) and did not vary seasonally. Nitrate and phosphate concentrations were generally low (0 to 3.68 mu M and 0.09 to 3 mu M for nitrate and phosphate respectively), whereas silicate was present in concentrations in the range 0.19 to 13 mu M. The phytoplankton community was dominated by diatoms, particularly at the inshore sites, as determined by a combination of microscopic and high-performance liquid chromatography (HPLC) pigment analyses. At the offshore sites the proportion of green flagellates increased. The cyanobacterium genus Trichodesmium and the diatom genera Chaetoceros, Rhizosolenia, Bacteriastrum and Thalassionema dominated the phytoplankton caught in 37 mu m mesh nets; however, in contrast to many other coastal areas studied worldwide there was no distinct species succession of the diatoms and only Trichodesmium showed seasonal changes in abundance. This reflects a stable phytoplankton community in waters without pulses of physical and chemical disturbances. These results are discussed in the context of the commercial prawn fishery in the Gulf of Carpentaria and the possible effect of phytoplankton on prawn larval growth and survival.

Carotenoid content and flesh color of selected banana cultivars growing in Australia.

Background: The problems of vitamin A deficiency and chronic diseases have emerged in recent years in some countries in the Micronesian region. These problems are associated with the dietary shift towards imported processed foods and lifestyle changes. Research in the Federated States of Micronesia indicates that yellow- and orange-fleshed banana cultivars contain significant levels of provitamin A carotenoids. Objective: To identify further banana cultivars that may be promoted to alleviate vitamin A deficiency among children and women and chronic disease problems among adults. Methods: Ripe fruit of banana cultivars growing in Australia (sourced mostly from a field research collection) were assessed for carotenoid content and flesh color. Ten cultivars with yellow or yellow/orange flesh color (including common cultivars of Southeast Asia and the Pacific Islands) were selected and compared with two cream-fleshed cultivars, including Williams, of the Cavendish group, the most commonly marketed banana worldwide. Carotenoid content was analyzed by high-performance liquid chromatography (HPLC). Flesh color was analyzed by HunterLab colorimetry. Results: The yellow/orange-fleshed Asupina (a Fe'i banana) contained the highest level (1,412 μg/100 g) of trans β-carotene, the most important provitamin A carotenoid, a level more than 20 times higher than that of Williams. All 10 yellow or yellow/orange-fleshed cultivars (Asupina, Kirkirnan, Pisang Raja, Horn Plantain, Pacific Plantain, Kluai Khai Bonng, Wain, Red Dacca, Lakatan, and Sucrier) had significant carotenoid levels, potentially meeting half or all of the estimated vitamin A requirements for a nonpregnant, nonlactating adult woman within normal consumption patterns. All were acceptable for taste and other attributes. The cream-fleshed cultivars had minimal carotenoid levels. There was a positive significant correlation between carotenoid content and deeper yellow/orange coloration indicators. Conclusions: These yellow- or yellow/orange-fleshed carotenoid-rich banana cultivars should be considered for promotion in order to alleviate vitamin A deficiency and chronic disease in susceptible target communities and to provide variety and enjoyment as exotic fruits in both developing and industrialized countries.

A rapid and sensitive method for the estimation of individual tRNA pools

A rapid and sensitive method is described to quantitatively compare tRNA pools for individual aminoacids in a single experiment. The procedure comprises of: (i) charging of total tRNA with a mixture of radiolabeled aminoacids, (ii) deacylation of the esterified tRNA with a volatile base and the recovery of the labeled aminoacid, (iii) derivatisation of the aminoacid with phenylisothiocyanate after mixing with excess of nonradioactive aminoacids, (iv) baseline separation of the phenylthiocarbamyl aminoacids by reverse phase high performance liquid chromatography monitored by A254nm and (v) quantitation of the radioactivity in individual aminoacid peaks. The radioactivity in the aminoacid peak corresponds to the quantity of the aminoacylated tRNA. The method has been successfully applied to quantitate the individual tRNA pools in the developing silk glands of Bombyx mori, a functionally adapted tissue which undergoes considerable variations in tRNA content. PSG, posterior silk gland; PITC, phenylisothiocyanate; DMAA, N,N-dimethyl-N-allylamine; APH, algal protein hydrolysate; ptc-, phenylthiocarbamyl; HPLC, high performance liquid chromatography.

Sleep deprivation and brain energy metabolism : in vivo studies in rats and humans

Sleep deprivation leads to increased subsequent sleep length and depth and to deficits in cognitive performance in humans. In animals extreme sleep deprivation is eventually fatal. The cellular and molecular mechanisms causing the symptoms of sleep deprivation are unclear. This thesis was inspired by the hypothesis that during wakefulness brain energy stores would be depleted, and they would be replenished during sleep. The aim of this thesis was to elucidate the energy metabolic processes taking place in the brain during sleep deprivation. Endogenous brain energy metabolite levels were assessed in vivo in rats and in humans in four separate studies (Studies I-IV). In the first part (Study I) the effects of local energy depletion on brain energy metabolism and sleep were studied in rats with the use of in vivo microdialysis combined with high performance liquid chromatography. Energy depletion induced by 2,4-dinitrophenol infusion into the basal forebrain was comparable to the effects of sleep deprivation: both increased extracellular concentrations of adenosine, lactate, and pyruvate, and elevated subsequent sleep. This result supports the hypothesis of a connection between brain energy metabolism and sleep. The second part involved healthy human subjects (Studies II-IV). Study II aimed to assess the feasibility of applying proton magnetic resonance spectroscopy (1H MRS) to study brain lactate levels during cognitive stimulation. Cognitive stimulation induced an increase in lactate levels in the left inferior frontal gyrus, showing that metabolic imaging of neuronal activity related to cognition is possible with 1H MRS. Study III examined the effects of sleep deprivation and aging on the brain lactate response to cognitive stimulation. No physiologic, cognitive stimulation-induced lactate response appeared in the sleep-deprived and in the aging subjects, which can be interpreted as a sign of malfunctioning of brain energy metabolism. This malfunctioning may contribute to the functional impairment of the frontal cortex both during aging and sleep deprivation. Finally (Study IV), 1H MRS major metabolite levels in the occipital cortex were assessed during sleep deprivation and during photic stimulation. N-acetyl-aspartate (NAA/H2O) decreased during sleep deprivation, supporting the hypothesis of sleep deprivation-induced disturbance in brain energy metabolism. Choline containing compounds (Cho/H2O) decreased during sleep deprivation and recovered to alert levels during photic stimulation, pointing towards changes in membrane metabolism, and giving support to earlier observations of altered brain response to stimulation during sleep deprivation. Based on these findings, it can be concluded that sleep deprivation alters brain energy metabolism. However, the effects of sleep deprivation on brain energy metabolism may vary from one brain area to another. Although an effect of sleep deprivation might not in all cases be detectable in the non-stimulated baseline state, a challenge imposed by cognitive or photic stimulation can reveal significant changes. It can be hypothesized that brain energy metabolism during sleep deprivation is more vulnerable than in the alert state. Changes in brain energy metabolism may participate in the homeostatic regulation of sleep and contribute to the deficits in cognitive performance during sleep deprivation.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Effect of phenolic-rich plant materials on protein and lipid oxidation reactions

The antioxidant activity of natural plant materials rich in phenolic compounds is being widely investigated for protection of food products sensitive to oxidative reactions. In this thesis plant materials rich in phenolic compounds were studied as possible antioxidants to prevent protein and lipid oxidation reactions in different food matrixes such as pork meat patties and corn oil-in water emulsions. Loss of anthocyanins was also measured during oxidation in corn oil-in-water emulsions. In addition, the impact of plant phenolics on amino acid level was studied using tryptophan as a model compound to elucidate their role in preventing the formation of tryptophan oxidation products. A high-performance liquid chromatography (HPLC) method with ultraviolet and fluorescence detection (UV-FL) was developed that enabled fast investigation of formation of tryptophan derived oxidation products. Byproducts of oilseed processes such as rapeseed (Brassica rapa L.), camelina (Camelina sativa) and soy meal (Glycine max L.) as well as Scots pine bark (Pinus sylvestris) and several reference compounds were shown to act as antioxidants toward both protein and lipid oxidation in cooked pork meat patties. In meat, the antioxidant activity of camelina, rapeseed and soy meal were more pronounced when used in combination with a commercial rosemary extract (Rosmarinus officinalis). Berry phenolics such as black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins showed potent antioxidant activity in corn oil-in-water emulsions toward lipid oxidation with and without β-lactoglobulin. The antioxidant effect was more pronounced in the presence of β-lactoglobulin. The berry phenolics also inhibited the oxidation of tryptophan and cysteine side chains of β-lactoglobulin. The results show that the amino acid side chains were oxidized prior the propagation of lipid oxidation, thereby inhibiting fatty acid scission. In addition, the concentration and color of black currant anthocyanins decreased during the oxidation. Oxidation of tryptophan was investigated in two different oxidation models with hydrogen peroxide (H2O2) and hexanal/FeCl2. Oxidation of tryptophan in both models resulted in oxidation products such as 3a-hydroxypyrroloindole-2-carboxylic acid, dioxindolylalanine, 5-hydroxy-tryptophan, kynurenine, N-formylkynurenine and β-oxindolylalanine. However, formation of tryptamine was only observed in tryptophan oxidized in the presence of H2O2. Pine bark phenolics, black currant anthocyanins, camelina meal phenolics as well as cranberry proanthocyanidins (Vaccinium oxycoccus) provided the best antioxidant effect toward tryptophan and its oxidation products when oxidized with H2O2. The tryptophan modifications formed upon hexanal/FeCl2 treatment were efficiently inhibited by camelina meal followed by rapeseed and soy meal. In contrast, phenolics from raspberry, black currant, and rowanberry (Sorbus aucuparia) acted as weak prooxidants. This thesis contributes to elucidating the effects of natural phenolic compounds as potential antioxidants in order to control and prevent protein and lipid oxidation reactions. Understanding the relationship between phenolic compounds and proteins as well as lipids could lead to the development of new, effective, and multifunctional antioxidant strategies that could be used in food, cosmetic and pharmaceutical applications.

Folates in rye: Determination and enhancement by food processing

The average daily intake of folate, one of the B vitamins, falls below recommendations among the Finnish population. Bread and cereals are the main sources of folate, rye being the most significant single source. Processing is a prerequisite for the consumption of whole grain rye; however, little is known about the effect of processing on folates. Moreover, data on the bioavailability of endogenous cereal folates are scarce. The aim of this study was to examine the variation in as well as the effect of fermentation, germination, and thermal processes on folate contents in rye. Bioavailability of endogenous rye folates was investigated in a four-week human intervention study. One of the objectives throughout the work was to optimise and evaluate analytical methods for determining folate contents in cereals. Affinity chromatographic purification followed by high-performance liquid chromatography (HPLC) was a suitable method for analysing cereal products for folate vitamers, and microbiological assay with Lactobacillus rhamnosus reliably quantified the total folate. However, HPLC gave approximately 30% lower results than the microbiological assay. The folate content of rye was high and could be further increased by targeted processing. The vitamer distribution of whole grain rye was characterised by a large proportion of formylated vitamers followed by 5-methyltetrahydrofolate. In sourdough fermentation of rye, the studied yeasts synthesized and lactic acid bacteria mainly depleted folate. Two endogenous bacteria isolated from rye flour were found to produce folate during fermentation. Inclusion of baker s yeast in sourdough fermentation raised the folate level so that the bread could contain more folate than the flour it was made of. Germination markedly increased the folate content of rye, with particularly high folate concentrations in hypocotylar roots. Thermal treatments caused significant folate losses but the preceding germination compensated well for the losses. In the bioavailability study, moderate amounts of endogenous folates in the form of different rye products and orange juice incorporated in the diet improved the folate status among healthy adults. Endogenous folates from rye and orange juice showed similar bioavailability to folic acid from fortified white bread. In brief, it was shown that the folate content of rye can be enhanced manifold by optimising and combining food processing techniques. This offers some practical means to increase the daily intake of folate in a bioavailable form.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Glycoalkaloid Content and Starch Structure in Solanum Species and Interspecific Somatic Potato Hybrids

When genome sections of wild Solanum species are bred into the cultivated potato (S. tuberosum L.) to obtain improved potato cultivars, the new cultivars must be evaluated for their beneficial and undesirable traits. Glycoalkaloids present in Solanum species are known for their toxic as well as for beneficial effects on mammals. On the other hand, glycoalkaloids in potato leaves provide natural protection against pests. Due to breeding, glycoalkaloid profile of the plant is affected. In addition, the starch properties in potato tubers can be affected as a result of breeding, because the crystalline properties are determined by the botanical source of the starch. Starch content and composition affect the texture of cooked and processed potatoes. In order to determine glycoalkaloid contents in Solanum species, simultaneous separation of glycoalkaloids and aglycones using reversed-phase high-performance liquid chromatography (HPLC) was developed. Clean-up of foliage samples was improved using a silica-based strong cation exchanger instead of octadecyl phases in solid-phase extraction. Glycoalkaloids alpha-solanine and alpha-chaconine were detected in potato tubers of cvs. Satu and Sini. The total glycoalkaloid concentration of non-peeled and immature tubers was at an acceptable level (under 20 mg/100 g of FW) in the cv. Satu, whereas concentration in cv. Sini was 23 mg/100 g FW. Solanum species (S. tuberosum, S. brevidens, S. acaule, and S. commersonii) and interspecific somatic hybrids (brd + tbr, acl + tbr, cmm + tbr) were analyzed for their glycoalkaloid contents using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The concentrations in the tubers of the brd + tbr and acl + tbr hybrids remained under 20 mg/100 g FW. Glycoalkaloid concentration in the foliage of the Solanum species was between 110 mg and 890 mg/100 g FW. However, the concentration in the foliage of S. acaule was as low as 26 mg/100 g FW. The total concentrations of brd + tbr, acl + tbr, and cmm + tbr hybrid foliages were 88 mg, 180 mg, and 685 mg/100 g FW, respectively. Glycoalkaloids of both parental plants as well as new combinations of aglycones and saccharides were detected in somatic hybrids. The hybrids contained mainly spirosolanes, and glycoalkaloid structures having no 5,6-double bond in the aglycone. Based on these results, the glycoalkaloid profiles of the hybrids may represent a safer and more beneficial spectrum of glycoalkaloids than that found in currently cultivated varieties. Starch nanostructure of three different cultivars (Satu, Saturna, and Lady Rosetta), a wild species S. acaule, and interspecific somatic hybrids were examined by wide-angle and small-angle X-ray scattering (WAXS, SAXS). For the first time, the measurements were conducted on fresh potato tuber samples. Crystallinity of starch, average crystallite size, and lamellar distance were determined from the X-ray patterns. No differences in the starch nanostructure between the three different cultivars were detected. However, tuber immaturity was detected by X-ray scattering methods when large numbers of immature and mature samples were measured and the results were compared. The present study shows that no significant changes occurred in the nanostructures of starches resulting from hybridizations of potato cultivars.

Concentrations of phthalates and DINCH metabolites in pooled urine from Queensland, Australia

Dialkyl phthalate esters (phthalates) are ubiquitous chemicals used extensively as plasticizers, solvents and adhesives in a range of industrial and consumer products. 1,2-Cyclohexane dicarboxylic acid, diisononyl ester (DINCH) is a phthalate alternative introduced due to a more favourable toxicological profile, but exposure is largely uncharacterised. The aim of this study was to provide the first assessment of exposure to phthalates and DINCH in the general Australian population. De-identified urine specimens stratified by age and sex were obtained from a community-based pathology laboratory and pooled (n = 24 pools of 100). Concentrations of free and total species were measured using online solid phase extraction isotope dilution high performance liquid chromatography tandem mass spectrometry. Concentrations ranged from 2.4 to 71.9 ng/mL for metabolites of di(2-ethylhexyl)phthalate, and from < 0.5 to 775 ng/mL for all other metabolites. Our data suggest that phthalate metabolites concentrations in Australia were at least two times higher than in the United States and Germany; and may be related to legislative differences among countries. DINCH metabolite concentrations were comparatively low and consistent with the limited data available. Ongoing biomonitoring among the general Australian population may help assess temporal trends in exposure and assess the effectiveness of actions aimed at reducing exposures.

Grain sorghum proteomics: integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants

Grain protein composition determines quality traits, such as value for food, feedstock, and biomaterials uses. The major storage proteins in sorghum are the prolamins, known as kafirins. Located primarily on the periphery of the protein bodies surrounding starch, cysteine-rich beta- and gamma-kafirins may limit enzymatic access to internally positioned alpha-kafirins and starch. An integrated approach was used to characterize sorghum with allelic variation at the kafirin loci to determine the effects of this genetic diversity on protein expression. Reversed-phase high performance liquid chromatography and lab-on-a-chip analysis showed reductions in alcohol-soluble protein in beta-kafirin null lines. Gel-based separation and liquid chromatography-tandem mass spectrometry identified a range of redox active proteins affecting storage protein biochemistry. Thioredoxin, involved in the processing of proteins at germination, has reported impacts on grain digestibility and was differentially expressed across genotypes. Thus, redox states of endosperm proteins, of which kafirins are a subset, could affect quality traits in addition to the expression of proteins.