Quantification of in vitro malodor generation by anionic surfactant-induced fluorescent sensor property of tryptophan

In this article, we report tuning of the sensory capability of an amino acid (tryptophan) in a biomimicking anionic micellar nano cage. It has been shown that anionic surfactant concentration dictates the sensing behavior of tryptophan toward body malodor component (butyric acid) generated by bacterial degradation of tributyrin. We have proposed a fluorescence quenching mechanism that is based on short-chain fatty acid (SCFA) proximity with tryptophan present at the micelle-water interface. Anionic surfactant-induced fluorescent sensor activity of tryptophan exhibits high sensitivity (detection limit up to 10 μM) and specific selectivity (toward SCFA, < C12) in aqueous solution. We also determined antibacterial efficacy of various zinc salts based on the sensory activity of tryptophan, which has been correlated with the established resazurin assay.     

Characterization of two different acyl carrier proteins in complex I from Yarrowia lipolytica

Acyl carrier proteins of mitochondria (ACPMs) are small (∼ 10 kDa) acidic proteins that are homologous to the corresponding central components of prokaryotic fatty acid synthase complexes. Genomic deletions of the two genes ACPM1 and ACPM2 in the strictly aerobic yeast Yarrowia lipolytica resulted in strains that were not viable or retained only trace amounts of assembled mitochondrial complex I, respectively. This suggested different functions for the two proteins that despite high similarity could not be complemented by the respective other homolog still expressed in the deletion strains. Remarkably, the same phenotypes were observed if just the conserved serine carrying the phosphopantethein moiety was exchanged with alanine. Although this suggested a functional link to the lipid metabolism of mitochondria, no changes in the lipid composition of the organelles were found. Proteomic analysis revealed that both ACPMs were tightly bound to purified mitochondrial complex I. Western blot analysis revealed that the affinity tagged ACPM1 and ACPM2 proteins were exclusively detectable in mitochondrial membranes but not in the mitochondrial matrix as reported for other organisms. Hence we conclude that the ACPMs can serve all their possible functions in mitochondrial lipid metabolism and complex I assembly and stabilization as subunits bound to complex I.     

Probing the amyloid-β(1-40) fibril environment with substituted tryptophan residues

A signature feature of Alzheimer’s disease is the accumulation of plaques, composed of fibrillar amyloid-β protein (Aβ), in the brain parenchyma. Structural models of Aβ fibrils reveal an extensive β-sheet network with a hydrophobic core extending throughout the fibril axis. In this study, phenylalanines in the Aβ(1-40) sequence were substituted with tryptophan residues at either position 4 (F4W) or 19 (F19W) to probe the fibril environment. The F4W substitution did not alter self-assembly kinetics, while the F19W change slightly lengthened the lag phase without hindering fibril formation. The tryptophan fluorescence of Aβ(1-40) F19W, but not Aβ(1-40) F4W, underwent a marked blue shift during fibril formation and this shift was temporally correlated with thioflavin T binding. Isolated Aβ(1-40) F19W fibrils exhibited the largest fluorescence blue shifts consistent with W19 insertion into the Aβ(1-40) fibril inner core and direct probing of the substantially hydrophobic environment therein.     

Development of a novel fluorometric assay for nitric oxide utilizing sesamol and its application to analysis of nitric oxide‐releasing drugs

Nitric oxide (NO) is related to various physiological effects as well as to numerous diseases caused by accentuation of NO production. Measurement of NO in cells and tissues is difficult as NO readily reacts with other molecules; furthermore, its half‐life as a radical is fleeting. Currently, many NO pharmaceuticals are marketed as therapeutic agents for ischemic disease. Consequently, the identification of NO radicals and determination of generation rate from pharmaceuticals is very important when the effect of the medicinal supply is estimated. In this study, we developed a fluorometric assay for NO employing sesamol (3,4‐methylenedioxyphenol) as a fluorometric substrate. Sesamol is converted to a fluorescent derivative (ex. 365 nm, em. 447 nm), which is dimmer in the presence of NO. The detection limit of NO with this method is 400 fmol; moreover, NO generated from drugs can be measured. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

Response of plasma and gastrointestinal melatonin,plasma cortisol and activity rhythms of European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) to dietary supplementation with tryptophan and melatonin

Melatonin is an effective antioxidant, immunostimulant, gonadal maturating regulator and antistress indoleamine that may be potentially useful for fish farmers. We have explored two possible ways of increasing plasma melatonin levels through the diet: direct melatonin supplementation (ME diet) and supplementation with the melatonin precursor tryptophan (TRP diet). To this end, a group of sea bass was fed a commercial diet (STD diet) at a regular time for 16 days, after which plasma, intestine, and bile samples were taken at four different time points: 120 min before, and 15, 180 and 480 min after feeding. Locomotor activity, intestinal and biliary melatonin, and plasma melatonin, serotonin and cortisol levels were measured. This same sampling process and analyses were also carried out after feeding sea bass TRP diet or ME diet for 1 week. Our results show that melatonin, but not tryptophan supplementation of the diet increases plasma, intestine and bile levels of melatonin. Plasma serotonin levels, on the other hand, were increased by dietary tryptophan, but not by melatonin, confirming the availability of supplemented tryptophan for serotonin synthesis. Both treatments were equally effective in reducing the high cortisol levels observed with the STD diet.     

HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization

We have characterized the viral RNA conformation in wild-type, protease-inactive (PR-) and SL1-defective (DeltaDIS) human immunodeficiency virus type 1 (HIV-1), as a function of the age of the viruses, from newly released to grown-up (>or=24 h old). We report evidence for packaging HIV-1 genomic RNA (gRNA) in the form of monomers in PR- virions, viral RNA rearrangement (not maturation) within PR- HIV-1, protease-dependent formation of thermolabile dimeric viral RNAs, a new form of immature gRNA dimer at about 5 h post virion release, and slow-acting dimerization signals in SL1-defective viruses. The rates of gRNA dimer formation were >or=3-fold and >or=10-fold slower in DeltaDIS and PR- viruses than in wild-type, respectively. Thus, the DIS, i.e. the palindrome in the apical loop of SL1, is a dimerization initiation signal, but its role can be masked by one or several slow-acting dimerization site(s) when grown-up SL1-inactive virions are investigated. Grown-up PR- virions are not flawless models for immature virions because gRNA dimerization increases with the age of PR- virions, indicating that the PR- mutation does not "freeze" gRNA conformation in a nascent primordial state. Our study is the first on gRNA conformation in newly released mutant or primate retroviruses. It shows for the first time that the packaged retroviral gRNA matures in more than one step, and that formation of immature dimeric viral RNA requires viral protein maturation. The monomeric viral RNAs isolated from budding HIV-1, as modeled by newly released PR- virions, may be seen as dimers that are much more fragile than thermolabile dimers.     

Tandem dimerization of the human p53 tetramerization domain stabilizes a primary dimer intermediate and dramatically enhances its oligomeric stability

Tetramerization of the human p53 tumor suppressor protein is required for its biological functions. However, cellular levels of p53 indicate that it exists predominantly in a monomeric state. Since the oligomerization of p53 involves the rate-limiting formation of a primary dimer intermediate, we engineered a covalently linked pair of human p53 tetramerization (p53tet) domains to generate a tandem dimer (p53tetTD) that minimizes the energetic requirements for forming the primary dimer. We demonstrate that p53tetTD self-assembles into an oligomeric structure equivalent to the wild-type p53tet tetramer and exhibits dramatically enhanced oligomeric stability. Specifically, the p53tetTD dimer exhibits an unfolding/dissociation equilibrium constant of 26 fM at 37 degrees C, or a million-fold increase in stability relative to the wild-type p53tet tetramer, and resists subunit exchange with monomeric p53tet. In addition, whereas the wild-type p53tet tetramer undergoes coupled (i.e. two-state) dissociation/unfolding to unfolded monomers, the p53tetTD dimer denatures via an intermediate that is detectable by differential scanning calorimetry but not CD spectroscopy, consistent with a folded p53tetTD monomer that is equivalent to the p53tet primary dimer. Given its oligomeric stability and resistance against hetero-oligomerization, dimerization of p53 constructs incorporating the tetramerization domain may yield functional constructs that may resist exchange with wild-type or mutant forms of p53.     

Cryptochrome 3 from Arabidopsis thaliana: structural and functional analysis of its complex with a folate light antenna

Cryptochromes are almost ubiquitous blue-light receptors and act in several species as central components of the circadian clock. Despite being evolutionary and structurally related with DNA photolyases, a class of light-driven DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs to the DASH-type subfamily. Its crystal structure determined at 1.9 Angstroms resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor 5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Angstroms to the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the photoreduction of FAD.