LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

Monepantel irreversibly binds to and opens Haemonchus contortus MPTL-1 and Caenorhabditis elegans ACR-20 receptors.

Monepantel is a recently developed anthelmintic with a novel mode of action. Parasitic nematodes with reduced sensitivity to monepantel have led to the identification of MPTL-1, a ligand-gated ion-channel subunit of the parasitic nematode Haemonchus contortus, as a potential drug target. Homomeric MPTL-1 channels reconstituted in Xenopus oocytes are gated by µM concentrations of betaine and mM concentrations of choline. Measurement of reversal potentials indicated that the channel has a similar conductance for Na(+) and K(+) ions and does not permeate Ca(2+). Concentrations of monepantel (amino-acetonitrile derivative [AAD]-2225) >0.1 μM, but not its inactive enantiomer AAD-2224, induced channel opening in an irreversible manner. Currents elicited by monepantel alone were larger than the maximal current amplitudes achieved with betaine or choline, making monepantel a superagonist. Currents elicited by betaine or choline were allosterically potentiated by nM concentrations of monepantel and to a much smaller degree by AAD-2224. We have also reconstituted the Caenorhabditis elegans homomeric ACR-20 receptor in Xenopus oocytes. The acr-20 sequence has higher similarity to mptl-1 than acr-23, the primary target for monepantel mode of action in C. elegans. The ACR-20 channel is gated similarly as MPTL-1. Monepantel, but not AAD-2224, was able to induce channel opening in an irreversible manner at similar concentrations as for MPTL-1. Interestingly, the allosteric potentiation measured in the presence of betaine was much smaller than in MPTL-1 receptors. Together, these results establish the mode of action of monepantel in H. contortus and contribute to our understanding of the mode of action of this anthelmintic.

Macrocyclic chelator-coupled gastrin-based radiopharmaceuticals for targeting of gastrin receptor-expressing tumours

PURPOSE: Diethylenetriamine-pentaacetic acid (DTPA)-coupled minigastrins are unsuitable for therapeutic application with the available beta-emitting radiometals due to low complex stability. Low tumour-to-kidney ratio of the known radiopharmaceuticals is further limiting their potency. We used macrocyclic chelators for coupling to increase complex stability, modified the peptide sequence to enhance radiolytic stability and studied tumour-to-kidney ratio and metabolic stability using (111)In-labelled derivatives. METHODS: Gastrin derivatives with decreasing numbers of glutamic acids were synthesised using (111)In as surrogate for therapeutic radiometals for in vitro and in vivo studies. Gastrin receptor affinities of the (nat)In-metallated compounds were determined by receptor autoradiography using (125)I-CCK as radioligand. Internalisation was evaluated in AR4-2J cells. Enzymatic stability was determined by incubating the (111)In-labelled peptides in human serum. Biodistribution was performed in AR4-2J-bearing Lewis rats. RESULTS: IC(50) values of the (nat)In-metallated gastrin derivatives vary between 1.2 and 4.8 nmol/L for all methionine-containing derivatives. Replacement of methionine by norleucine, isoleucine, methionine-sulfoxide and methionine-sulfone resulted in significant decrease of receptor affinity (IC(50) between 9.9 and 1,195 nmol/L). All cholecystokinin receptor affinities were >100 nmol/L. All (111)In-labelled radiopeptides showed receptor-specific internalisation. Serum mean-life times varied between 2.0 and 72.6 h, positively correlating with the number of Glu residues. All (111)In-labelled macrocyclic chelator conjugates showed higher tumour-to-kidney ratios after 24 h (0.37-0.99) compared to (111)In-DTPA-minigastrin 0 (0.05). Tumour wash out between 4 and 24 h was low. Imaging studies confirmed receptor-specific blocking of the tumour uptake. CONCLUSIONS: Reducing the number of glutamates increased tumour-to-kidney ratio but resulted in lower metabolic stability. The properties of the macrocyclic chelator-bearing derivatives make them potentially suitable for clinical purposes.

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K-m = 0.1-0.3 mM), lower for Pro (K-m = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K-m = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.

The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Comparison of C-11 methionine and C-11 choline for PET imaging of brain metastases: a prospective pilot study

The aim of the study was the comparison of C-11 methionine (MET) and C-11 choline (CHO) in the positron emission tomography (PET) imaging of brain metastases in correlation to the histopathology findings in stereotactic biopsy.

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

Experimental folate and vitamin B12 deficiency does not alter bone quality in rats

Hyperhomocysteinemia (HHCY) has been linked to fragility fractures and osteoporosis. Folate and vitamin B(12) deficiencies are among the main causes of HHCY. However, the impact of these vitamins on bone health has been poorly studied. This study analyzed the effect of folate and vitamin B(12) deficiency on bone in rats. We used two groups of rats: a control group (Co, n = 10) and a vitamin-deficient group (VitDef, n = 10). VitDef animals were fed for 12 wk with a folate- and vitamin B(12)-free diet. Co animals received an equicaloric control diet. Tissue and plasma concentrations of homocysteine (HCY), S-adenosyl-homocysteine (SAH), and S-adenosyl-methionine (SAM) were measured. Bone quality was assessed by biomechanical testing (maximum force of an axial compression test; F(max)), histomorphometry (bone area/total area; B.Ar./T.Ar.], and the measurement of biochemical bone turnover markers (osteocalcin, collagen I C-terminal cross-laps [CTX]). VitDef animals developed significant HHCY (Co versus VitDef: 6.8 +/- 2.7 versus 61.1 +/- 12.8 microM, p < 0.001) that was accompanied by a high plasma concentration of SAH (Co versus VitDef: 24.1 +/- 5.9 versus 86.4 +/- 44.3 nM, p < 0.001). However, bone tissue concentrations of HCY, SAH, and SAM were similar in the two groups. Fmax, B.Ar./T.Ar., OC, and CTX did not differ between VitDef and Co animals, indicating that bone quality was not affected. Folate and vitamin B(12) deficiency induces distinct HHCY but has no effect on bone health in otherwise healthy adult rats. The unchanged HCY metabolism in bone is the most probable explanation for the missing effect of the vitamin-free diet on bone.

Chromium supplementation and substitution of barley grain with corn: Effects on performance and lactation in periparturient dairy cows

Thirty-two multiparous Holstein cows were used to investigate the effects of chromium-l-methionine (Cr-Met) supplementation and dietary grain source on performance and lactation during the periparturient period. Cows were fed a total mixed ration consisting of either a barley-based diet (BBD) or a corn-based diet (CBD) from 21 d before anticipated calving through 28 d after calving. The Cr-Met was supplemented at dosages of 0 or 0.08 mg of Cr/kg of metabolic body weight. The study was designed as a randomized complete block design with 2 (Cr-Met levels) x 2 (grain sources) factorial arrangement. There was no Cr effect on prepartum dry matter intake (DMI) or postpartum DMI, body weight (BW), net energy balance, and whole tract apparent digestibility of nutrients. Prepartum DMI as a percentage of BW tended to increase with Cr-Met. Supplemental Cr-Met tended to increase milk yield whereas milk protein percentage decreased. Pre- and postpartum DMI, BW, net energy balance, milk yield, and milk composition were not affected by substituting ground barley with ground corn. The addition of Cr-Met increased prepartum DMI and tended to increase postpartum DMI of the BBD but not the CBD. The change in prepartum DMI was smaller when the BBD was supplemented with Cr-Met but remained unchanged when the CBD was supplemented with Cr-Met. Yields of crude protein and total solids in milk and prepartum digestibility of DM and organic matter tended to increase when Cr-Met was added to the BBD but remained unchanged when added to the CBD. Periparturient cows failed to respond to the grain source of the diet, whereas they showed greater response in milk yield to diets supplemented with Cr-Met. In conclusion, the present results demonstrate that the beneficial effect of Cr-Met supplementation during the periparturient period to improve feed intake may depend on the grain source of the diet.

Oxidative Stress in Hypobaric Hypoxia and Influence on Vessel-Tone Modifying Mediators

Increased pulmonary artery pressure is a well-known phenomenon of hypoxia and is seen in patients with chronic pulmonary diseases, and also in mountaineers on high altitude expedition. Different mediators are known to regulate pulmonary artery vessel tone. However, exact mechanisms are not fully understood and a multimodal process consisting of a whole panel of mediators is supposed to cause pulmonary artery vasoconstriction. We hypothesized that increased hypoxemia is associated with an increase in vasoconstrictive mediators and decrease of vasodilatators leading to a vasoconstrictive net effect. Furthermore, we suggested oxidative stress being partly involved in changement of these parameters. Oxygen saturation (Sao2) and clinical parameters were assessed in 34 volunteers before and during a Swiss research expedition to Mount Muztagh Ata (7549 m) in Western China. Blood samples were taken at four different sites up to an altitude of 6865 m. A mass spectrometry-based targeted metabolomic platform was used to detect multiple parameters, and revealed functional impairment of enzymes that require oxidation-sensitive cofactors. Specifically, the tetrahydrobiopterin (BH4)-dependent enzyme nitric oxide synthase (NOS) showed significantly lower activities (citrulline-to-arginine ratio decreased from baseline median 0.21 to 0.14 at 6265 m), indicating lower NO availability resulting in less vasodilatative activity. Correspondingly, an increase in systemic oxidative stress was found with a significant increase of the percentage of methionine sulfoxide from a median 6% under normoxic condition to a median level of 30% (p<0.001) in camp 1 at 5533 m. Furthermore, significant increase in vasoconstrictive mediators (e.g., tryptophan, serotonin, and peroxidation-sensitive lipids) were found. During ascent up to 6865 m, significant altitude-dependent changes in multiple vessel-tone modifying mediators with excess in vasoconstrictive metabolites could be demonstrated. These changes, as well as highly significant increase in systemic oxidative stress, may be predictive for increase in acute mountain sickness score and changes in Sao2.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

Hydrolytic behaviour of mono-and dithiolato-bridged dinuclear arene ruthenium complexes and their interactions with biological ligands

The hydrolysis and the reactivity of two dinuclear p-cymene ruthenium monothiolato complexes, [(η6-p-MeC6H4Pri)2Ru2Cl2(µ-Cl)(µ-S-m-9-B10C2H11)] (1) and [(η6-p-MeC6H4Pri)2¬Ru2Cl2(µ-Cl)¬(µ-S¬CH2-p-C6H4-NO2)] (2), and of two dinuclear p-cymene ruthenium dithiolato complexes, [(η6-p-MeC6H4Pri)2Ru2(µ-SCH2CH2Ph)2Cl2] (3) and [(η6-p-Me¬C6H4¬Pri)2¬Ru2(S¬CH2¬C6H4-p-O¬Me)2¬Cl2] (4) towards amino acids, nucleotides, and a single-stranded DNA dodecamer were studied using NMR and mass spectrometry. In aqueous solutions at 37 °C, the monothiolato com¬plexes 1 and 2 undergo rapid hydrolysis, irrespective of the pH value, the predominant species in D2O/acetone-d6 solution at equilibrium being the neutral hydroxo complexes [(η6-p-Me¬C6H4¬Pri)2Ru2(OD)2(µ-OD)(µ-SR)]. The dithiolato complexes 3 and 4 are stable in water under acidic conditions, but undergo slow hydrolysis under neutral and basic conditions. In both cases, the cationic hydroxo complexes [(η6-p-MeC6H4Pri)2Ru2(µ-SR)2¬(OD)¬(CD3CN)]+ are the only spe¬cies observed in D2O/CD3CN at equilibrium. Surprisingly, no adducts are observed upon addition of an excess of L-methionine or L-histidine to the aqueous solutions of the complexes. Upon addition of an excess of L-cysteine, on the other hand, 1 and 2 form the unusual cationic trithiolato complexes [(η6-p-MeC6H4Pri)2¬Ru2{µ-SCH2CH(NH2)COOH}2(µ-SR)]+ containing two bridging cysteinato li¬gands, while 3 and 4 yield cationic trithiolato complexes [(η6-p-MeC6H4Pri)2Ru2[µ-SCH2CH¬(NH2)COOH](µ-SR)2]+ containing one bridging cysteinato ligand. A representative of catio¬nic trithiolato complexes containing a cysteinato bridge of this type, [(η6-p-MeC6H4Pri)2¬Ru2[µ-S¬CH2CH(NH2)COOH](µ-SCH2-p-C6H4-But)2]+ (6) could be synthesised from the di¬thiolato complex [(η6-p-Me¬C6H4¬Pri)2-Ru2(S¬CH2¬C6H4-p-But)2Cl2] (5), isolated as the tetra¬fluo¬ro¬borate salt and fully characterised. Moreover, the mono- and dithiolato complexes 1 - 4 are inert toward nucleotides and DNA, suggesting that DNA is not a target of cytotoxic thiolato-bridged arene ruthenium complexes. In contrast to the trithiolato complexes, monothiolato and dithio¬lato complexes hydrolyse and react with L-cysteine. These results may have im¬portant implications for the mode of action of thiolato-bridged dinuclear arene ruthenium drug candidates, and suggest that their modes of action are different to those of other arene ruthenium complexes.