Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria

Phenylketonuria, an autosomal recessive Mendelian disorder, is one of the most common inborn errors of metabolism. Although currently treated by diet, many suboptimal outcomes occur for patients. Neuropathological outcomes include cognitive loss, white matter abnormalities, and hypo- or demyelination, resulting from high concentrations and/or fluctuating levels of phenylalanine. High phenylalanine can also result in competitive exclusion of other large neutral amino acids from the brain, including tyrosine and tryptophan (essential precursors of dopamine and serotonin). This competition occurs at the blood brain barrier, where the L-type amino acid transporter, LAT1, selectively facilitates entry of large neutral amino acids. The hypothesis of these studies is that certain non-physiological amino acids (NPAA; DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), α-aminoisobutyrate (AIB), and α-methyl-aminoisobutyrate (MAIB)) would competitively inhibit LAT1 transport of phenylalanine (Phe) at the blood-brain barrier interface. To test this hypothesis, Pah-/- mice (n=5, mixed gender; Pah+/-(n=5) as controls) were fed either 5% NL, 0.5% NB, 5% AIB or 3% MAIB (w/w 18% protein mouse chow) for 3 weeks. Outcome measurements included food intake, body weight, brain LNAAs, and brain monoamines measured via LCMS/MS or HPLC. Brain Phe values at sacrifice were significantly reduced for NL, NB, and MAIB, verifying the hypothesis that these NPAAs could inhibit Phe trafficking into the brain. However, concomitant reductions in tyrosine and methionine occurred at the concentrations employed. Blood Phe levels were not altered indicating no effect of NPAA competitors in the gut. Brain NL and NB levels, measured with HPLC, verified both uptake and transport of NPAAs. Although believed predominantly unmetabolized, NL feeding significantly increased blood urea nitrogen. Pah-/-disturbances of monoamine metabolism were exacerbated by NPAA intervention, primarily with NB (the prototypical LAT inhibitor). To achieve the overarching goal of using NPAAs to stabilize Phe transport levels into the brain, a specific Phe-reducing combination and concentration of NPAAs must be found. Our studies represent the first in vivo use of NL, NB and MAIB in Pah-/- mice, and provide proof-of-principle for further characterization of these LAT inhibitors. Our data is the first to document an effect of MAIB, a specific system A transport inhibitor, on large neutral amino acid transport.

Functionalization of carbon nanotubes using aminobenzene acids and electrochemical methods. Electroactivity for the oxygen reduction reaction

Functionalized carbon nanotubes (CNTs) using three aminobenzene acids with different functional groups (carboxylic, sulphonic, phosphonic) in para position have been synthesized through potentiodynamic treatment in acid media under oxidative conditions. A noticeable increase in the capacitance for the functionalized carbon nanotubes mainly due to redox processes points out the formation of an electroactive polymer thin film on the CNTs surface along with covalently bonded functionalities. The CNTs functionalized using aminobenzoic acid rendered the highest capacitance values and surface nitrogen content, while the presence of sulfur and/or phosphorus groups in the aminobenzene structure yielded a lower functionalization degree. The oxygen reduction reaction (ORR) activity of the functionalized samples was similar to that of the parent CNTs, independently of the functional group present in the aminobenzene acid. Interestingly, a heat treatment in N2 atmosphere with a very low O2 concentration (3125 ppm) at 800 °C of the CNTs functionalized with aminobenzoic acid produced a material with high amounts of surface oxygen and nitrogen groups (12 and 4% at., respectively), that seem to modulate the electron-donor properties of the resulting material. The onset potential and limiting current for ORR was enhanced for this material. These are promising results that validates the use of electrochemistry for the synthesis of novel N-doped electrocatalysts for ORR in combination with adequate heat treatments.

Three-dimensional hydrogen-bonded structures in the guanidinium salts of the monocyclic dicarboxylic acids rac-trans-cyclohexane-1,2-dicarboxylic acid (2:1, anhydrous), isophthalic acid (1:1, monohydrate) and terephthalic acid (2:1, trihydrate).

The structures of bis(guanidinium)rac-trans-cyclohexane-1,2-dicarboxylate, 2(CH6N3+) C8H10O4- (I), guanidinium 3-carboxybenzoate monohydrate CH6N3+ C8H5O4- . H2O (II) and bis(guanidinium) benzene-1,4-dicarboxylate trihydrate, 2(CH6N3+) C8H4O4^2- . 3H2O (III) have been determined and the hydrogen bonding in each examined. All three compounds form three-dimensional hydrogen-bonded framework structures. In anhydrous (I), both guanidinium cations give classic cyclic R2/2(8) N--H...O,O'(carboxyl) and asymmetric cyclic R1/2(6) hydrogen-bonding interactions while one cation gives an unusual enlarged cyclic interaction with O acceptors of separate ortho-related carboxyl groups [graph set R2/2(11)]. Cations and anions also associate across inversion centres giving cyclic R2/4(8) motifs. In the 1:1 guanidinium salt (II), the cation gives two separate cyclic R1/2(6) interactions, one with a carboxyl O-acceptor, the other with the water molecule of solvation. The structure is unusual in that both carboxyl groups give short inter-anion O...H...O contacts, one across a crystallographic inversion centre [2.483(2)\%A], the other about a two-fold axis of rotation [2.462(2)\%A] with a half-occupancy hydrogen delocalized on the symmetry element in each. The water molecule links the cation--anion ribbon structures into a three-dimensional framework. In (III), the repeating molecular unit comprises a benzene-1,4-dicarboxylate dianion which lies across a crystallographic inversion centre, two guanidinium cations and two water molecules of solvation (each set related by two-fold rotational symmetry), and a single water molecule which lies on a two-fold axis. Each guanidinium cation gives three types of cyclic interactions with the dianions: one R^1^~2~(6), the others R2/3(8) and R3/3(10) (both of these involving the water molecules), giving a three-dimensional structure through bridges down the b cell direction. The water molecule at the general site also forms an unusual cyclic R2/2(4) homodimeric association across an inversion centre [O--H...O, 2.875(2)\%A]. The work described here provides further examples of the common cyclic guanidinium cation...carboxylate anion hydrogen-bonding associations as well as featuring other less common cyclic motifs.

Hydrogen bonding in the anhydrous 1:1 proton-transfer compounds of isonipecotamide with nitro-substituted benzoic acids: the salts of the three isomeric mononitrobenzoic acids and of 3,5-dinitrobenzoic acid

The structures of the anhydrous 1:1 proton-transfer compounds of isonipecotamide (piperidine-4-carboxamide) with the three isomeric mononitro-substituted benzoic acids and 3,5-dinitrobenzoic acid, namely 4-carbamoylpiperidinium 2-nitrobenzoate (I), 4-carbamoylpiperidinium 3-nitrobenzoate (II), 4-carbamoylpiperidinium 4-nitrobenzoate (III), (C6H13N2O+ C7H4NO4-) and 4-carbamoylpiperidinium 3,5-dinitrobenzoate (IV) (C6H13N2O+ C7H5N2O6-)respectively, have been determined at 200 K. All salts form hydrogen-bonded structures: three-dimensional in (I), two-dimensional in (II) and (III) and one-dimensional in (IV). Featured in the hydrogen bonding of three of these [(I), (II) and (IV)] is the cyclic head-to-head amide--amide homodimer motif [graph set R2/2~(8)] through a duplex N---H...O association, the dimer then giving structure extension via either piperidinium or amide H-donors and carboxylate-O and in some examples [(II) and (IV)], nitro-O atom acceptors. In (I), the centrosymmetric amide-amide homodimers are expanded laterally through N-H...O hydrogen bonds via cyclic R2/4(8) interactions forming ribbons which extend along the c cell direction. These ribbons incorporate the 2-nitrobenzoate cations through centrosymmetric cyclic piperidine N-H...O(carboxyl) associations [graph set R4/4(12)], giving inter-connected sheets in the three-dimensional structure. In (II) in which no amide-amide homodimer is present, duplex piperidinium N-H...O(amide) hydrogen-bonding homomolecular associations [graph set R2/2(14)] give centrosymmetric head-to-tail dimers. Structure extension occurs through hydrogen-bonding associations between both the amide H-donors and carboxyl and nitro O-acceptors as well as a three-centre piperidinium N-H...O,O'(carboxyl) cyclic R2/1(4) association giving the two-dimensional network structure. In (III), the centrosymmetric amide-amide dimers are linked through the two carboxyl O-atom acceptors of the anions via bridging piperidinium and amide N-H...O,O'...H-N(amide) hydrogen bonds giving the two-dimensional sheet structure which features centrosymmetric cyclic R4/4(12) associations. In (IV), the amide-amide dimer is also centrosymmetric with the dimers linked to the anions through amide N-H...O(nitro) interactions. The piperidinium groups extend the structure into one-dimensional ribbons via N-H...O(carboxyl) hydrogen bonds. The structures reported here further demonstrate the utility of the isonipecotamide cation in molecular assembly and highlight the efficacy of the cyclic R2/2(8) amide-amide hydrogen-bonding homodimer motif in this process and provide an additional homodimer motif type in the head-to-tail R2/2(14) association.

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

Introduction. There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation. Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Aim. To investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. Methods. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 in the absence or presence of (-)-bupranolol. Results. The potencies (pEC50) of (-)-CGP12177 were β2AR (9.24 ± 0.14, n = 5), β1(V230I)AR (9.07 ± 0.07, n = 10), β1(β2TMDV)AR (8.86 ± 0.10, n = 15), β1(R222Q)AR (8.09 ± 0.29, n = 6), β1AR (8.00 ± 0.11, n = 11). The affinities (pKB) of (-)-bupranolol were β2AR (9.82 ± 0.52, n = 5), β1(V230I)AR (7.64 ± 0.12, n = 8), β1(β2TMV)AR (8.06 ± 0.17, n = 8), β1(R222Q)AR (7.33 ± 0.23, n = 5), β1AR (7.23 ± 0.23, n = 5). Discussion. The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR). The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion TMDV of the β1AR contributes in part to the low affinity binding site of β1AR.

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation (reviewed Kaumann and Molenaar, 2008). Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site (Baker et al 2002) and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Our aim was to investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 or (-)-isoprenaline in the absence or presence of (-)-bupranolol. _______________________________________________________________________ (-)-CGP 12177 (-)-Bupranolol affinity (pKB) pEC50 vs (-)-CGP 12177 vs (-)-isoprenaline _______________________________________________________________________ β1AR 8.00 ± 0.11 (11) 7.23 ± 0.23 (5) 9.52 ± 0.28 (5) β2AR (high density) 9.24 ± 0.14 (5) 9.82 ± 0.52 (8) xPaulxxxxxxx β2AR (low density) no effect β1(β2TMV)AR 8.86 ± 0.10 (15) 8.06 ± 0.17 (8) 9.08 ± 0.22 (6) β1(V230I)AR 9.07 ± 0.07 (10) 7.64 ± 0.12 (8) 9.36 ± 0.28 (9) β1(R222Q)AR 8.09 ± 0.29 (6) 7.33 ± 0.23 (5) 9.36 ± 0.08 (6) β1(V230A)AR 7.59 ± 0.09 (6) 7.32 ± 0.24 (4) 8.62 ± 0.18 (5) _______________________________________________________________________ The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR) but not β2AR. The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol at β1AR and mutants was higher when determined with (-)-isoprenaline than with (-)-CGP 12177. The affinity of (-)-bupranolol determined against (-)-CGP 12177 was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion V230 of the β1AR contributes in part to the low affinity binding site of β1AR. Baker JG, Hall IP, Hill SJ (2002). Pharmacological characterization of CGP12177 at the human β2-adrenoceptor. Br J Pharmacol 137, 400−408 Kaumann AJ, Molenaar P (2008) The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology. Pharmacol Ther 118, 303-336

Proton-transfer compounds of isnipecotamide with the aromatic dicarboxylic acids 4-nitrophthalic, 4,5-dichlorophthalic, 5-nitroisophthalic and terephthalic acid

The structures of the 1:1 proton-transfer compounds of isonipecotamide (4-piperidinecarboxamide) with 4-nitrophthalic acid, 4-carbamoylpiperidinium 2-carboxy-4-nitrobenzoate, C6H13N2O8+ C8H4O6- (I), 4,5-dichlorophthalic acid, 4-carbamoylpiperidinium 2-carboxy-4,5-dichlorobenzoate, C6H13N2O8+ C8H3Cl2O4- (II) and 5-nitroisophthalic acid, 4-carbamoylpiperidinium 3-carboxy-5-nitrobenzoate, C6H13N2O8+ C8H4O6- (III) as well as the 2:1 compound with terephthalic acid, bis(4-carbamoylpiperidinium)benzene-1,2-dicarboxylate dihydrate, 2(C6H13N2O8+) C8H4O42- . 2H2O (IV)have been determined at 200 K. All salts form hydrogen-bonded structures, one-dimensional in (II) and three-dimensional in (I), (III) and (IV). In (I) and (III) the centrosymmetric R2/2(8) cyclic amide-amide association is found while in (IV) several different types of water-bridged cyclic associations are present [graph sets R2/4(8), R3/4(10), R4/4(12), R3/3(18) and R4/6(22)]. The one-dimensional structure of (I), features the common 'planar' hydrogen 4,5-dichlorophthalate anion together with enlarged cyclic R3/3(13) and R3/4(17) associations. With the structures of (I) and (III) the presence of head-to-tail hydrogen phthalate chain substructures is found. In (IV) head-to-tail primary cation-anion associations are extended longitudinally into chains through the water-bridged cation associations and laterally by piperidinium N-H...O(carboxyl) and water O-H...O(carboxyl) hydrogen bonds. The structures reported here further demonstrate the utility of the isonipecotamide cation as a synthon for the generation of stable hydrogen-bonded structures. An additional example of cation--anion association with this cation is also shown in the asymmetric three-centre piperidinium N-H...O,O'(carboxyl) interaction in the first-reported structure of a 2:1 isonipecotamide-carboxylate salt.

rac-cis-Cyclohexaane-1,2-dicarboxylic acid-isoquinoline (1/1)

In the structure of the title molecular adduct C8H12O4 . C9H7N, the two species are interlinked through a carboxylic acid-isoquinoline O-H...N hydrogen bond, these molecular pairs then inter-associate through the second acid group of the cis-cyclohexane-1,2-dicarboxylic acids, forming a classic centrosymmetric cyclic head-to-head carboxylic acid--carboxyl O---H...O hydrogen-bonding association [graph set R^2^~2~(8)], giving a zero-dimensional structure.

Optimisation of process parameters for the removal of hydroxycinnamic acids in sugar solutions

Colour is one of the most important parameters in sugar quality and its presence in raw sugar plays a key role in the marketing strategy of sugar industries worldwide. This study investigated the degradation of a mixture of colour precursors using the Fenton oxidation process. These colour precursors are caffeic acid, p–coumaric acid and ferulic acid, which are present in cane juice. Results showed that with a Fe(II) to H2O2 molar ratio of 1:15 in an aqueous system at 25 °C, 77% of the total phenolic acid content was removed at pH 4.72. However, in a synthetic juice solution which contained 13 mass % sucrose (35 °C, pH 5.4), only 60% of the total phenolic acid content was removed.

Cyclic Imide and Open-Chain Amide Carboxylic Acid Derivatives from the Facile Reaction of cis-Cyclohexane-1,2-dicarboxylic Anhydride with Three Substituted Anilines

The structures of the compounds from the reaction of cis-cyclohexane-1,2-dicarboxylic anhydride with 4-chloroaniline [rac-N-(4-chlorophenyl)-2-carboxycycloclohexane-1-carboxamide] (1), 4-bromoaniline [2-(4-bromophenyl)-perhydroisoindolyl-1,3-dione] (2) and 3-hydroxy-4-carboxyaniline (5-aminosalicylic acid) [2-(3-hydroxy-4-carboxyphenyl)-perhydroisoindolyl-1,3-dione] (3) have been determined at 200 K. Crystals of the open-chain amide carboxylic acid 1 are orthorhombic, space group Pbcn, with unit cell dimensions a = 20.1753(10), b = 8.6267(4), c = 15.9940(9) Å, and Z = 8. Compounds 2 and 3 are cyclic imides, with 1 monoclinic having space group P21 and cell dimensions a = 11.5321(3), b = 6.7095(2), c = 17.2040(5) Å, β = 102.527(3)o. Compound 3 is orthorhombic with cell dimensions a = 6.4642(3), b = 12.8196(5), c = 16.4197(7) Å. Molecules of 1 form hydrogen-bonded cyclic dimers which are extended into a two-dimensional layered structure through amide-group associations: 3 forms into one-dimensional zigzag chains through carboxylic acid…imide O-atom hydrogen bonds, while compound 2 is essentially unassociated. With both cyclic imides 2 and 3, disorder is found which involves the presence of partial enantiomeric replacement of the cis-cyclohexane-1,2-substituted ring systems.

Omega-3 polyunsaturated fatty acids in the treatment of kidney disease

After more than 25 years of published investigation, including randomized controlled trials, the role of omega-3 polyunsaturated fatty acids in the treatment of kidney disease remains unclear. In vitro and in vivo experimental studies support the efficacy of omega-3 polyunsaturated fatty acids on inflammatory pathways involved with the progression of kidney disease. Clinical investigations have focused predominantly on immunoglobulin A (IgA) nephropathy. More recently, lupus nephritis, polycystic kidney disease, and other glomerular diseases have been investigated. Clinical trials have shown conflicting results for the efficacy of omega-3 polyunsaturated fatty acids in IgA nephropathy, which may relate to varying doses, proportions of eicosapentaenoic acid and docosahexaenoic acid, duration of therapy, and sample size of the study populations. Meta-analyses of clinical trials using omega-3 polyunsaturated fatty acids in IgA nephropathy have been limited by the quality of available studies. However, guidelines suggest that omega-3 polyunsaturated fatty acids should be considered in progressive IgA nephropathy. Omega-3 polyunsaturated fatty acids decrease blood pressure, a known accelerant of kidney disease progression. Well-designed, adequately powered, randomized, controlled clinical trials are required to further investigate the potential benefits of omega-3 polyunsaturated fatty acids on the progression of kidney disease and patient survival.