Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.     

N 6 -( 2 -isopentenyl) adenosine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli

An enzyme has been partially purified from Escherichia coli which catalyzes in vitro the transfer of the Δ²-isopentenyl group from Δ²-isopentenyl pyrophosphate to an adenosine residue in Mycoplasma sp. (Kid) tRNA. The product of the reaction is N⁶-(Δ²-isopentenyl) adenosine, which is known to be absent in this Mycoplasma tRNA. The enzyme has an approximate molecular weight of 55,000 daltons, requires reduced sulfhydryl groups and a divalent metal ion for full activity, and is specific for tRNA.     

Contents of Macroelements and Growth of Sweet Cherry Rootstock in vitro

Rhododendron shoot regeneration was accomplished using either flower explants (each consisting of ovary with pedicel) of Rhododendron cvs. Nova Zembla and Irina or leaves isolated from in vitro grown Rhododendron catawbiense Michx. Multiple shoot tip clumps were obtained on Anderson's medium containing 0.5 to 1.5 mg dm⁻³ thidiazuron (TDZ) in combination with 12 to 15 mg dm⁻³ N⁶-[2-isopentenyl]adenine (2iP) and 1 to 3 mg dm⁻³ indole-3-butyric acid (IBA). After 16 weeks on the regeneration media, explants with shoot tip clumps were transferred for shoot elongation to Anderson's medium with 3 mg dm⁻³ 2iP. Two months later, the shoots have reached 5 to 40 mm in length and were fit for subcultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

A dye-binding assay for measurement of the binding of Cu(II) to proteins

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to α-synuclein of ∼1 × 10⁹ M⁻¹ was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).     

Purification of the nitrogenase proteins from Clostridium pasteurianum

A method is described for the purification of the nitrogenase proteins from Clostridium pasteurianum by two polyethylene glycol precipitations and chromatography on columns of DEAE-cellulose, Sephadex G-100 and Sephadex G-200. The Mo-Fe protein and the Fe protein have been purified 70–80-fold from the crude extract, and they appear essentially pure when tested by anaerobic polyacrylamide gel electrophoresis.     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

Dinitrogen fixation by field grown soybeans: statistical analysis of variations in δ15N and proposed sampling procedureand proposed sampling procedure

Summary This paper reports a field study for the assessment of the variation in¹⁵N natural abundance method to estimate nitrogen fixation. For two successive years (1979 and 1980) at the same site, distribution of the variable δ¹⁵N in populations of nodulating and non-nodulating soybeans (Glycine max) has been studied. In 1980, the population structure was studied in order to detect a neighbour effect which could explain bimodal trend observed in the distribution. There was no evidence for such an effect. A sampling procedure is proposed: with 15 to 30 nodulating plants chosen randomly, a confidence interval of ±0.5δ can be obtained, while less than 10 non-nodulating plants are sufficient to achieve a very good precision. Some other aspects are studied: effect of pooling plants which considerably reduce the experimental work; the use of a non leguminous reference plant is possible. All our results show that fixing capacity introduces major variability in the δ¹⁵N measured in the plants, while the absence of fixation leads to a homogenous population, so the percentage of fixed N can be evaluated with a rather good precision. The higher the reference value, the better the accuracy of this evaluation, for a given precision of a δ¹⁵N measurement.     

Characterization of caged compounds binding to proteins by NMR spectroscopy

Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested.     

Synthesis,characterization, and studies on DNA binding of a new Mg(II) complex with N<Superscript>1</Superscript>,N<Superscript>8</Superscript>-bis(1-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine

A new Mg(II) complex of MgL(NO3)2 (here L = N(1),N(8)-bis(1-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine) has been synthesized and characterized. The interactions between the Mg(II) complex and calf thymus DNA has been investigated using UV spectra, fluorescent spectra, viscosity, thermal denaturation, and molecular modeling. The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is non-classical electrostatic action and the complex can cleave pBR322 DNA.     

Facile synthesis of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N 6-ethenoadenosine nucleosides

Facile synthetic methods of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N ⁶-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthetic products were isolated and purified by preparative HPLC and their structures were confirmed by¹H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.     

Two distinct oxysterol binding protein-related proteins in the parasitic protist Cryptosporidium parvum (Apicomplexa)

Two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) have been identified from the parasitic protist Cryptosporidium parvum (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans including Plasmodium, Toxoplasma, and Eimeria possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.     

N6-methyl adenosine in siRNA evades immune response without reducing RNAi activity

Abstract

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N⁶-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N⁶-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.     

Convenient and Efficient Syntheses of Oligodeoxyribonucleotides Containing O 6-(Carboxymethyl)Guanine and O 6-(4-Oxo-4-(3-Pyridyl)Butyl)Guanine

O ⁶-(carboxymethyl)guanine (O ⁶-CMG) and O ⁶-(4-oxo-4-(3-pyridyl)butyl)guanine (O ⁶-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O ⁶-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O ⁶-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O ⁶-CMG and O ⁶-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O ⁶-pobG and O ⁶-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental file.]     

Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K _d 6.7 × 10^–7 M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavinbinding site of the enzyme, apo-L-aspartate oxidase has been reacted with N⁶-(6-carboxyhexyl)-FAD Succinimidoester. The structural characterization of the resulting N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/ monomer. Residue Lys38 was identified as the target of the covalent modification. N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.     

Calcium binding by spinach stromal proteins

Calcium binding to spinach (Spinacia oleracea L.) stromal proteins was examined by dual-wavelength spectrophotometry using the metallochromic indicator tetramethylmurexide. The data are consistent with the existence of at least two, probably independent, classes of binding sites. The total number of binding sites varied between 90–155 nmol·mg^–1 protein with average binding constants of 1.1–2.7·mM^–1. Both Mg²⁺ and La³⁺ inhibited calcium binding competitively, with average inhibitor constants of 0.26·mM^–1 and 39.4·mM^–1, respectively; an increase in the potassium concentration up to 50 mM had no effect. In a typical experiment a decrease in pH (7.8 to 7.1) resulted in a decrease in the total number of calcium binding sites from 90 to 59 nmol·mg^–1 protein, but in an increase of the average affinity from 2.7 to 4.5·mM^–1. Calculations, using these data and those of Gross and Hess (1974, Biochim. Biophys. Acta 339, 334–346) for binding site I of washed thylakoid membranes, showed that the free-Ca²⁺ concentration in the stroma under dark conditions, pH 7.1, is higher than under light conditions, pH 7.8. The physiological relevance of the observed calcium binding by stromal proteins is discussed.Abbreviations Ca _b ²⁺ bound calcium - Ca _f ²⁺ free calcium     

The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Resolving temporal variation in vertebrate diets using naturally occurring stable isotopes

Assessments of temporal variation in diets are important for our understanding of the ecology of many vertebrates. Ratios of naturally occurring stable isotopes in animal tissues are a combination of the source elements and tissue specific fractionation processes, and can thus reveal dietary information. We review three different approaches that have been used to resolve temporal diet variation through analysis of stable isotopes. The most straightforward approach is to compare samples from the same type of tissue that has been sampled over time. This approach is suited to address either long or short-term dietary variation, depending on sample regime and which tissue that is sampled. Second, one can compare tissues with different metabolic rates. Since the elements in a given tissue have been assimilating during time spans specific to its metabolic rate, tissues with different metabolic rates will reflect dietary records over different periods. Third, comparisons of sections from tissues with progressive growth, such as hair, feathers, claws and teeth, will reveal temporal variation since these tissues will retain isotopic values in a chronological order. These latter two approaches are mainly suited to address questions regarding intermediate and short-term dietary variation. Knowledge of tissue specific metabolic rates, which determine the molecular turnover for a specific tissue, is of central importance for all these comparisons. Estimates of isotopic fractionation between source and measured target are important if specific hypotheses regarding the source elements are addressed. Estimates of isotopic fractionation, or at least of differences in fractionation between tissues, are necessary if different tissues are compared. We urge for more laboratory experiments aimed at improving our understanding of differential assimilation of dietary components, isotopic fractionation and metabolic routing. We further encourage more studies on reptiles and amphibians, and generally more studies utilizing multiple tissues with different turnover rates.