Mode of action of N,N′-diphenylurea: The isolation and identification of the cytokinins in the transfer RNA from tobacco callus grown in the presence of N,N′-diphenylurea

Summary The tRNA from cytokinin-dependent tobacco callus (Nicotiana tabacum) grown on mineral medium containing N,N-diphenylurea as the source of cytokinin was found to contain 3 cytokinin-active ribonucleosides. The 2 ribonucleosides present in the largest amounts were identified conclusively by their chromatographic properties, ultra-violet and low-resolution mass spectra as the naturally-occurring cytokinins 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine and 6-(3-methyl-2-butenylamino)-9--ribofuranosylpurine. A third ribonucleoside, present in smaller amounts, was identified as another naturally-occurring cytokinin 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9--D-ribofuranosylpurine on the basis of its chromatographic behaviour. No evidence was found to associate the mode of action of the non-purine cytokinin, N,N-diphenylurea, with tRNA.Abbreviation DPU N,N-diphenylurea     

Chemistry of the N′-Oxides of Nicotine and Myosmine

Nicotine-N′-oxide (II) was purified to give a crystalline form which has m.p. 170~171°C, and . The reaction of nicotine-N′-oxide with acetic anhydride afforded a good yield of 1′-(3-pyridyl)-4′-(N′-acetylmethylamino)-l′-propanone (V) which was hydrolyzed to give pseudoöxynicotine (III). Nicotine-N′-oxide (II) either with acetyl chloride or with benzoyl chloride, under similar conditions, furnished pseudoöxynicotine (III) without giving the corresponding acyl compound as an intermediate. 2′-Methyl-6′-(3-pyridyl)-tetrahydro-l′,2′-oxazine (XII) rearranged from nicotine-N′-oxide reacted neither with acetic anhydride nor acetyl chloride. Reduction of the oxime (IV) of pseudoöxynicotine dihydrochloride gave dl-l′-amino-l′-(3-pyridyl)-4′-methyl-aminobutane (VII) as a main product. The hydrazone (VIII) of pseudoöxynicotine dihydrochloride subjected to a modification of the Wolf-Kischner reaction, was reduced to yield dihydrometanicotine (IX). The pyrolysis of N′-methylmyosmine (IV) gave N′-methylnicotinamide (XI) and nicotyrine (X) in low yields. The presence of N′-methylmyosmine in the autoxidation mixture of nicotine was also established. Oxidation of nornicotine (XIV) with hydrogen peroxide furnished myosmine-N′-oxide (XV) whose identity was established by its chemical and physical properties. This oxide, on pyrolysis, gave nornicotyrine (XVII) and myosmine (XVI).     

N,N,N′,N′-tetramethyl-p-phenylenediamine initiates the appearance of a well-resolved I peak in the kinetics of chlorophyll fluorescence rise in isolated thylakoids

Addition of N,N,N′,N′-tetramethyl-p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J–I–P phase of fluorescence rise and greatly retarded the I–P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence (F_v) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

Phagocidal Activity of the Hydrolyzate of Acetal Derivatives of Oxidized Spermine and Its N,N′-Dimethyl Analog

The hydrolysis of acetal derivatives of oxidized spermine, N, N′-bis (3, 3-diethoxypropyl)-1, 4-diaminobutane, and its N, N′-dimethyl analog with acid was practically complete within 1 hr. During the hydrolysis of these compounds, no detectable amounts of acrolein were formed in the reaction mixture. While the hydrolyzate of the acetal derivative of oxidized spermine potently inactivated bacteriophage T¹, that of the N, N′-dimethyl analog had little phagocidal activity.     

Measurement of the apparent diffusion coefficient of N‐butane in soil

Measurements of in‐soil diffusion coefficients and the application of an appropriate diffusional model can allow for a more accurate prediction of soil gas concentrations and movement to locate subterranean contamination of volatile materials. The present study was undertaken to measure and evaluate the “apparent in‐soil diffusion coefficient”; for n‐butane through soil columns under non‐steady‐state conditions. The term “apparent in‐soil diffusion coefficient”; refers to a numerical coefficient that primarily describes the movement of the material by diffusion but also contains effects due to other mechanisms (e.g., adsorption and solubility).

Six test columns were evaluated at three soil porosity levels ranging from 0.30 to 0.43 and at two column temperature conditions, nominally 18°C and 7°C. Soil columns measured 25.4 cm in diameter by 84 cm in height and contained a moist sand/silt/clay mixture. The numerical range for the apparent in‐soil diffusion coefficients for n‐butane was 0.447 × 10^‐3cm²/s to 0.561 × 10^‐3cm²/s. The lower coefficient values were associated with lower soil porosity levels and cooler column conditions.     

Structure of the vesicular stomatitis virus N⁰-P complex

Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N_Δ21) and a peptide of 60 amino acids (P₆₀) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N⁰). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.     

Does the combination of optimal substitutions at the C²-, N⁵- and N⁸-positions of the pyrazolo-triazolo-pyrimidine scaffold guarantee selective modulation of the human A₃ adenosine receptors?

In an attempt to study the optimal combination of a phenyl ring at the C(2)-position and different substituents at the N(5)- and N(8)-positions towards the selective modulation of human A(3) adenosine receptors (hA(3)AR), we synthesized a new series of 2-para-(un)substituted-phenyl-pyrazolo-triazolo-pyrimidines bearing either a methyl or phenylethyl at N(8) and chains of variable length at N(5). Through biological evaluation, it was found that the majority of the compounds had good affinities towards the hA(3)AR in the low nanomolar range. Compound 16 possessed the best hA(3)AR affinity and selectivity profile (K(i)hA(3)=1.33 nM; hA(1)/hA(3)=4880; hA(2A)/hA(3)=1100) in the present series of 2-(substituted)phenyl-pyrazolo-triazolo-pyrimidine derivatives. In addition to pharmacological characterization, a molecular modeling investigation on these compounds further elucidated the effect of different substituents at the pyrazolo-triazolo-pyrimidine scaffold on affinity and selectivity to hA(3)AR.     

Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM

Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF(3)(-)-CheY, a phosphorylated CheY (CheY approximately P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY approximately P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF(3)(-)-CheY-FliM(16) complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg(2+)-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF(3)(-)-activated CheY. This structure makes clear the active role that the beta4-alpha4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

On the inhibition of HIV-1 protease by hydrazino-ureas displaying the N→CO interaction

To create novel HIV-1 protease (HIV PR) inhibitors, we have extended our investigations of the N→CO interaction as a moiety that reproduces electrostatic properties of the transition state of peptidolysis. Consequently, we prepared a series of compounds with an unusual hydrazino-urea core. In polar protic media, these adopt solely a cyclic constitution displaying the interaction on one side of the molecule while offering a urea moiety on the opposite side meant to hydrogen-bond with the enzyme flaps. Each inhibitor candidate was obtained via a key series of three synthetic steps employing carbonyl-di-imidazole (CDI). It was thus possible to efficiently fuse two independent building blocks, a hydrazine and a protected aminoaldehyde in a convergent manner. NMR and UV analysis proved that all compounds, when dissolved in polar protic media, existed exclusively in the cyclic constitution exhibiting the N→CO interaction. In total, five inhibitor candidates were tested with HIV PR for their potency. The one carrying the least bulk in peripheral substituents showed the highest activity. Its very low molecular weight (365 g/mol) holds great promise for future improvements in affinity without violating Lipinski’s rule of remaining within the limit of 500 g/mol.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

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Study of the urinary and faecal excretion of N ε-carboxymethyllysine in young human volunteers

The dietary habits of the adolescent population with a high intake of snack and fast foods mean that they consume a high rate of which in turn leads to the development of different degenerative disorders. There are few studies available on MRP absorption and metabolism. We investigated the effects of a MRP-high and a MRP-low diet on carboxymethyllysine (CML) intake and excretion in 11-14 years adolescent males. In a 2-period crossover trial, 20 healthy subjects were randomly assigned to two groups. The first group consumed the MRP-low diet for 2 weeks, observed a 40-day washout period, and then consumed the MRP-high diet for 2 weeks. The second group received the diets in the reverse order. Subjects collected urine and faeces on the last 3 days of each dietary period. The consumption of the MRP-high diet led to a higher CML input (P < 0.05) (11.28 vs. 5.36 mg/day CML for MRP-high and -low diet, respectively). In parallel, the faecal excretion was also greater (P < 0.05) (3.52 vs. 1.23 mg/day CML, respectively) and proportional to the dietary intake. The urinary elimination of CML was not increased significantly when the MRP-high diet was consumed compared to consumption of the MRP-low diet, and was not proportional to the dietary exposure of CML. In conclusion it was shown that CML absorption and faecal excretion were highly influenced by dietary CML levels. Since the compound has long-term effects on health, an excessive intake deserves attention, especially in a population nutritionally at risk as adolescents.     

Effect of N,N′-bis (alkyldimethyl)-α,ω-alkanediammonium dibromides on bacteria of the genusclostridium

Antibacterial effect of 17 ammonium compounds of the type of N,N′-bis(alkyldimethyl)-α,ω-alkanediammonium dibromides was tested on anaerobically sporulating bacteria of the genusClostridium. A sizable antibacterial activity was displayed by five N,N′-bis(alkyldimethyl)-1,6-hexanediammonium dibromides and by four N,N′-bis(decyldimethyl)-α,ω-alkanediammonium dibromides. These compounds exhibited activity higher than, or comparable with, that of the reference standards Ajatin and Septonex. The maximum antibacterial activity was found in compounds whose alkyl chain contained 9–12 carbon atoms. Compounds with a lower number of carbon atoms in the chain (less than 8) exhibited a low activity.     

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

The impact of N^α-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N^α-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N^α-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and β-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and β-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N^α-acetyltransferase.The multisubunit and ribosome-associated protein N^α-acetyltransferases (NATs)1 are omnipresent enzyme complexes that catalyze the transfer of the acetyl moiety from acetyl-CoA to the primary α-amines of N termini of nascent proteins (1–3). As up to 50 to 60% of yeast proteins and 80 to 90% of human proteins are modified in this manner, N^α-acetylation is a widespread protein modification in eukaryotes (4–7), and the pattern of modification has remained largely conserved throughout evolution (4, 8). NATs belong to a subfamily of the Gcn5-related N-acetyltransferase superfamily of N-acetyltransferases, additionally encompassing the well-studied histone acetyltransferases that are implicated in epigenetic imprinting.In yeast and humans, three main NAT complexes, NatA, NatB, and NatC were found to be responsible for the majority of N^α-terminal acetylations (1). The NatA complex, responsible for cotranslational N^α-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini, is composed of two main subunits, the catalytic subunit Naa10p (previously known as Ard1p) and the auxiliary subunit Naa15p (previously known as Nat1p/NATH) (9–11). Furthermore, a third catalytic subunit Naa50p (previously known as Nat5)—an acetyltransferase shown to function in chromosome cohesion and segregation (12–14)—was found to physically interact with the NatA complex of yeast (2), fruit fly (12), and human (15). Recently, human Naa50p (hNaa50p) was reported to display lysine or N^ε-acetyltransferase as well as NAT activity (16), the latter was defined as NatE activity (16). Interestingly, the chaperone-like, Huntingtin interacting protein HYPK, identified as a novel stable interactor of human NatA, was functionally implicated in the N-terminal acetylation of an in vivo NatA substrate, demonstrating that NAT complex formation and composition may have an overall influence on the observed (degree of) N^α-acetylation (17). Further, subunits of the human NatA complex have been coupled to cancer-related processes and differentiation, with altered subunit expression reported in papillary thyroid carcinoma, neuroblastoma, and retinoic acid induced differentiation. Furthermore, the NatA catalytic subunit was found to be implicated in processes such as hypoxia-response and the β-catenin pathway (18, 19). Of note is that in line with the differential localization patterns of the individual NatA subunits (9, 13, 20, 21), other data indicate that these subunits might well exert NatA-independent enzymatic functions (13, 22, 23). Given that a significant fraction of hNaa10p and hNaa15p are nonribosomal (9), and given the multitude of postulated post-translational in vivo N-acetylation events recently reported (24–26), these observations argue in favor of the existence of NAT complexes and/or catalytic NAT-subunits acting post-translationally.Similar to NatA, the NatB and NatC complexes, composed of the catalytic subunit Naa20p or Naa30p and the auxiliary subunits Naa25p or Naa35p and Naa38p respectively, are conserved from yeast to higher eukaryotes concerning their subunit composition as well as their substrate specificity. Both these complexes display activity toward methionine-starting N termini, with NatB preferring acidic residues as well as Asn and Gln at P2′-sites², whereas NatC prefers hydrophobic amino acid residues at substrate P2′-sites (1, 27, 28).N^α-acetylation affects various protein functions such as localization, activity, association, and stability (29, 30). Only recently a more generalized function of protein N^α-acetylation in generating so-called N-terminal degrons marking proteins for removal was put forward (31). The lack of mouse models in addition to the fact that (combined) knockdown of individual components of N^α-acetyltransferases only marginally affect the overall N^α-acetylation status (4) have so far hampered the molecular characterization of the substrate specificity profile of (yet uncharacterized) NATs. To date, all eukaryote N^α-acetylation events are assumed to be catalyzed by the five known NATs (32). However, an additional level of complexity is imposed by the fact that in contrast to yeast, higher eukaryotes express multiple splice variants of various NAT subunits as well as paralogs thereof (33, 34), further implicating that a specific NAT''s substrate specificity might be altered in this way, in addition to the possible existence of substrate redundancy. Moreover, regulation of substrate specificity and stability of NAT activity can be imposed by differential complex formation and post-translational modifications including phosphorylation, auto-acetylation, and specific proteolytic cleavage of the catalytic subunits (9, 16, 17). As such, a detailed understanding of the substrate specificity of NATs, and the regulation thereof, could help unravel the physiological substrate repertoires as well as the associated physiological roles of NATs in the normal and the disease state.The specificity of N^α-acetyltransferases and their endogenous substrates were originally studied by two-dimensional-PAGE: N^α-acetylation neutralizes the N-terminal positive charge, resulting in an altered electrophoretic protein migration during isoelectric focusing (35–38). Recently, this altered biophysical property was also exploited to enrich for protein N-termini using low pH strong cation exchange (SCX) chromatography (24, 39). As an example, SCX prefractionation combined with N-terminal combined fractional diagonal chromatography, a targeted proteomics technology negatively selecting for protein N-terminal peptides, stable isotope labeling of amino acids in cell culture, and amino-directed modifiers (40), was used to study the in vivo substrate repertoires of human as well as yeast NatA (4).Nevertheless, the various methods reported today to study in detail N^α-terminal acetylation and thus the specificities of different NATs make use of a limited and therefore somewhat biased set of synthesized peptide substrates and comprise the rather laborious detection of radioactive acetylated products as well as enzyme-coupled methods quantifying acetyl-CoA conversion. Because (proteome-derived) peptide libraries have been used extensively to study epitope mapping (41), protein-protein interactions (42), protein modifications such as phosphorylation (43), and proteolysis (44, 45), as well as for determining the substrate specificity of the N^α-deblocking peptide deformylase (46), we reckoned that the development of an oligopeptide-based acetylation assay should allow for more comprehensive screening of NAT-like activities. We here report on the development of a peptide-based method to systematically screen for the in vitro sequence specificity profile of individual NATs as well as endogenous NAT complexes. In summary, SCX enriched, N^α-free peptide libraries, derived from natural proteomes build up the peptide substrate pool. And, upon incubation, NAT N^α-acetylated peptides are enriched by a second SCX fractionation step, resulting in a positive selection of NAT-specific peptide substrates. By use of this proteome-derived peptide library approach, we here delineated (differences in) the specificity profiles of hNaa50p and hNaa10p as isolated hNatA components, as well as of assayed their combined activity when in their native hNatA complex.