N-Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease

Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.

Identification of a novel polymorphism in the 3'UTR of the L-arginine transporter gene SLC7A1 : contribution to hypertension and endothelial dysfunction

Background— Endothelial dysfunction because of reduced nitric oxide bioavailability is a key feature of essential hypertension. We have found that normotensive siblings of subjects with essential hypertension have impaired endothelial function accompanied by altered arginine metabolism.

Methods and Results— We have identified a novel C/T polymorphism in the 3′UTR of the principal arginine transporter, solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 gene (SLC7A1). The minor T allele significantly attenuates reporter gene expression (P<0.01) and is impaired in its capacity to form DNA-protein complexes (P<0.05). In 278 hypertensive subjects the frequency of the T allele was 13.3% compared with 7.6% in 498 normotensive subjects (P<0.001). Moreover, the overall genotype distribution observed in hypertensives differed significantly from that in normotensives (P<0.001). To complement these studies, we generated an endothelial-specific transgenic mouse overexpressing l-arginine transporter SLC7A1. The Slc7A1 transgenic mice exhibited significantly enhanced responses to the endothelium-dependent vasodilator acetylcholine (−log EC50 for wild-type versus Slc7A1 transgenic: 6.87±0.10 versus 7.56±0.13; P<0.001). This was accompanied by elevated production of nitric oxide by isolated aortic endothelial cells.

Conclusions— The present study identifies a key, functionally active polymorphism in the 3′UTR of SLC7A1. As such, this polymorphism may account for the apparent link between altered endothelial function, l-arginine, and nitric oxide metabolism and predisposition to essential hypertension.

Structural classification of proteins through amino acid sequence using interval type-2 fuzzy logic system

This paper introduces a new multi-output interval type-2 fuzzy logic system (MOIT2FLS) that is automatically constructed from unsupervised data clustering method and trained using heuristic genetic algorithm for a protein secondary structure classification. Three structure classes are distinguished including helix, strand (sheet) and coil which correspond to three outputs of the MOIT2FLS. Quantitative properties of amino acids are used to characterize the twenty amino acids rather than the widely used computationally expensive binary encoding scheme. Amino acid sequences are parsed into learnable patterns using a local moving window strategy. Three clustering tasks are performed using the adaptive vector quantization method to derive an equal number of initial rules for each type of secondary structure. Genetic algorithm is applied to optimally adjust parameters of the MOIT2FLS with the purpose of maximizing the Q3 measure. Comprehensive experimental results demonstrate the strong superiority of the proposed approach over the traditional methods including Chou-Fasman method, Garnier-Osguthorpe-Robson method, and artificial neural network models.

Changes in the amino acid profiles during embryonic development of the blacklip abalone (Haliotis Rubra)

Changes in the total amino acid (TAA) and the free amino acid (FAA) contents during embryonic development, through newly spawned eggs, to pre-settled larvae of blacklip abalone (Haliotis rubra) are described. The TAA (protein bound + free) and the FAA contents increased prior to hatching but decreased towards settlement, but the changes were not always significant between different stages of development. Threonine, arginine, lysine and leucine accounted for nearly 50 % of the total essential amino acids (TEAA) in all developmental stages. The mean FAA content of newly spawned eggs was 262.8 ± 28.2 pmol·ind^–1 and accounted for 11.5 ± 8.3 % of the TAA. Free essential amino acid (FEAA) content increased significantly as development progressed (P < 0.05), in which threonine, arginine and lysine accounted for over 63 % of this pool. In all developmental stages, the FAA pool was dominated by the non-essential amino acids taurine + proline which accounted for 79.5 % of the total. Generally, the FAA accounted for between 10 to 15 % of the TAA in the different developmental stages of blacklip abalone. All evidence appears to indicate that in blacklip abalone the energy requirements during early ontogeny are mostly met with from the lipid reserves, and that there is a tendency to conserve amino acids until pre-settlement.

Plasma amino acid response after ingestion of different whey protein fractions

Background and objectives The digestion rate of proteins and subsequent absorption of amino acids can independently modulate protein metabolism. The objective of the present study was to examine the blood amino acid response to whey protein isolate (WPI), β-lactoglobulin-enriched WPI, hydrolysed WPI and a flavour-identical control.

Methods Eight healthy adults (four female, four male) were recruited (mean±standard error of the mean: age, 27.0±0.76 years; body mass index, 23.2±0.8 kg/cm2) and after an overnight fast consumed 500 ml of each drink, each containing 25g protein, in a cross-over design. Blood was taken at rest and then every 15 min for 2 h post ingestion.

Results Ingesting the β-lactoglobulin-enriched WPI drink resulted in significantly greater plasma leucine concentrations at 45-120 min and significantly greater branched-chain amino acid concentrations at 60-105 min post ingestion compared with hydrolysed WPI. No differences were observed between WPI and β-lactoglobulin-enriched WPI, and all protein drinks resulted in elevated blood amino acids compared with flavour-identical control.

Conclusions In conclusion, whole proteins resulted in a more rapid absorption of leucine and branched-chain amino acid into the blood compared with the hydrolysed molecular form of whey protein.

Using polyatomic primary ions to probe an amino acid and a nucleic base in water ice

In this study on pure water ice, we show that protonated water species [H₂O]_nH⁺ are more prevalent than (H₂O)_n⁺ ions after bombardment by Au+ monoatomic and Au₃⁺ and C₆₀⁺ polyatomic projectiles. This data also reveals significant differences in water cluster yields under bombardment by these three projectiles. The amino acid alanine and the nucleic base adenine in solution have been studied and have been shown to have an effect on the water cluster ion yields observed using an Au₃⁺ ion beam.

Substituted naphthoquinones as novel amino acid sensitive reagents for the detection of latent fingermarks on paper surfaces

In this paper, we present our preliminary studies into naphthoquinones as novel reagents for the detection of latent fingermarks on paper. Latent fingermarks deposited on paper substrates were treated with solutions of selected naphthoquinones in ethyl acetate/HFE-7100, with subsequent heating. The selected compounds were 1,4-dihydroxy-2-naphthoic acid, 1,2-naphthoquinone-4-sulfonate, 2-methoxy-1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone. All of the tested compounds yielded purple-brown visible fingermarks, which also exhibited photoluminescence when illuminated with a high intensity filtered light source at 555nm and viewed through red goggles. Indirect heat using an oven at 150 ◦C for 1 h was found to be superior to direct heat with an iron, which while providing faster development lead to increased levels of background colouration. Luminescence spectrophotometry revealed differences in photoluminescence characteristics for fingermarks developed with the different naphthoquinones, with excitation over the range 530–590 nm. Luminescence spectrophotometry of developed lysine, glycine and serine spots on paper was used to confirm that the naphthoquinones were reacting with amino acids in the latent fingermark.

Organotin(IV) complexes derived from proteinogenic amino acid: Synthesis, structure and evaluation of larvicidal efficacy on Anopheles stephensi mosquito larvae

Five new organotin(IV) complexes of composition [Bz2SnL1]n (1), [Bz3SnL1HH2O] (2), [Me2SnL2H2O] (3), [Me2SnL3] (4) and [Bz3SnL3H]n (5) (where L1 = (2S)-2-([(E)-(4-hydroxypentan-2-ylidene)]amino)-4-methylpentanoate, L2 = (rac)-2-([(E)-1-(2-hydroxyphenyl)methylidene]amino)-4-methylpentanoate and L3 = (2S)- or (rac)-2-([(E)-1-(2-hydroxyphenyl)ethylidene]amino)-4-methylpentanoate) were synthesized and characterized using 1H NMR, 13C NMR, 119Sn NMR and infrared spectroscopic techniques. The crystal structure of 2 reveals a distorted trigonal-bipyramidal geometry around the tin atom where the oxygen atoms of the carboxylate ligand and a water ligand occupy the axial positions, while the three benzyl ligands are located at the equatorial positions. On the other hand, the analogous derivative of enantiopure L3H (5) consists of polymeric chains, in which the ligand-bridged tin atoms adopt the same trans-Bz3SnO2 trigonal-bipyramidal configuration and are now coordinated to a phenolic oxygen atom instead of H2O. In 2, the OH hydrogen of the ketoimine substituent has moved to the nearby nitrogen atom while in the salicylidene derivative 5, the OH is located almost midway between the phenolic oxygen atom and the nitrogen atom of the C=N group. For the dibenzyltin derivative 1, a polymeric chain structure is observed as a result of a long intermolecular SnO bond involving the exocyclic carbonyl oxygen atom from the tridentate ligand of a neighbouring tin-complex unit. The tin atom in this complex has distorted octahedral coordination geometry. In contrast, the racemic dimethyltin(IV) complexes 3 and 4 display discrete monomeric structures with a distorted octahedral- and trigonal-bipyramidal geometry, respectively. The structures show that the coordination mode of the Schiff base ligand depends primarily on the number of bulky benzyl ligands (R) at the tin atom, as indeed found in the structures of related complexes where R = phenyl. With three bulky R groups, the tridentate chelating O,N,O coordination mode is preferred, whereas with fewer or less bulky R ligands, only the carboxylate and hydroxy groups are involved, which leads to polymers. Larvicidal efficacies of two of the new tribenzyltin(IV) complexes (2 and 5) were assessed on the second larval instar of Anopheles stephensi mosquito larvae and compared with two triphenyltin(IV) analogues, [Ph3SnL1H]n and [Ph3SnL3H]n. The results demonstrate that the compounds containing Sn-Ph ligands are more effective than those with Sn-Bz ligands.

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

The last five amino acid residues at the C-terminus of PRK1 /KKN is essential for full lipid responsiveness

PRK1/PKN is a member of the protein kinase C (PKC) superfamily of serine/threonine protein kinases. Despite its important role as a RhoA effector, limited information is available regarding how this kinase is regulated. We show here that the last seven amino acid residues at the C-terminus is dispensable for the catalytic activity of PRK1 but is critical for the in vivo stability of this kinase. Surprisingly, the intact hydrophobic motif in PRK1 is dispensable for 3-phosphoinositide-dependent kinase-1 (PDK-1) binding and phosphorylation of the activation loop, as the PRK1-Δ940 mutant lacking the last two residues of the hydrophobic motif and the last 5 residues at the C-terminus interacts with PDK-1 in vivo and has a similar specific activity as the wild-type protein. We also found that the last four amino acid residues at the C-terminus of PRK1 is critical for the full lipid responsiveness as the PRK1-Δ942 deletion mutant is no longer activated by arachidonic acid. Our data suggest that the very C-terminus in PRK1 is critically involved in the control of the catalytic activity and activation by lipids. Since this very C-terminal segment is the least conserved among members of the PKC superfamily, it would be a promising target for isozyme-specific pharmaceutical interventions.

Conjugated linoleic acid suppresses myogenic gene expression in a model of human muscle cell inflammation

Proinflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha}, contribute to muscle wasting in inflammatory disorders, where TNF{alpha} acts to regulate myogenic genes. Conjugated linoleic acid (CLA) has shown promise as an antiproliferative and antiinflammatory agent, leading to its potential as a therapeutic agent in muscle-wasting disorders. To evaluate the effect of CLA on myogenesis during inflammation, human primary muscle cells were grown in culture and exposed to varying concentrations of TNF{alpha} and the cis-9, trans-11 and trans-10, cis-12 CLA isomers. Expression of myogenic genes (Myf5, MyoD, myogenin, and myostatin) and the functional genes creatine kinase (CK) and myosin heavy chain (MHC IIx) were measured by real-time PCR. TNF{alpha} significantly downregulated MyoD and myogenin expression, whereas it increased Myf5 expression. These changes corresponded with a decrease in both CK and MHC IIx expression. Both isomers of CLA mimicked the inhibitory effect of TNF{alpha} treatment on MyoD and myogenin expression, whereas myostatin expression was diminished in the presence of both isomers of CLA either alone or in combination with TNF{alpha}. Both isomers of CLA decreased CK and MHC IIx expression. These findings demonstrate that TNF{alpha} can have specific regulatory effects on myogenic genes in primary human muscle cells. A postulated antiinflammatory role of CLA in myogenesis appears more complex, with an indication that CLA may have a negative effect on this process.

Interacting amino acid preferences of 3D pattern pairs at the binding sites of transient and obligate protein complexes

To assess the physico-chemical characteristics of protein-protein interactions, protein sequences and overall structural folds have been analyzed previously. To highlight this, discovery and examination of amino acid patterns at the binding sites defined by structural proximity in 3-dimensional (3D) space are essential. In this paper, we investigate the interacting preferences of 3D pattern pairs discovered separately in transient and obligate protein complexes. These 3D pattern pairs are not necessarily sequence-consecutive, but each residue in two groups of amino acids from two proteins in a complex is within certain °A threshold to most residues in the other group. We develop an algorithm called AA-pairs by which every pair of interacting proteins is represented as a bipartite graph, and it discovers all maximal quasi-bicliques from every bipartite graph to form our 3D pattern pairs. From 112 and 2533 highly conserved 3D pattern pairs discovered in the transient and obligate complexes respectively, we observe that Ala and Leu is the highest occuring amino acid in interacting 3D patterns of transient (20.91%) and obligate (33.82%) complexes respectively. From the study on the dipeptide composition on each side of interacting 3D pattern pairs, dipeptides Ala-Ala and Ala-Leu are popular in 3D patterns of both transient and obligate complexes. The interactions between amino acids with large hydrophobicity difference are present more in the transient than in the obligate complexes. On contrary, in obligate complexes, interactions between hydrophobic residues account for the top 5 most occuring amino acid pairings.