The cleavage site for the restriction endonuclease EcoRV is 5′-GAT/ATC-3′

The cleavage site for the restriction endonuclease EcoRV has been found to be 5′-GAT/ATC-3′, rather than 5′-GATAT/C-3′ as reported earlier by Kholmina et al. [Dokl. Akad. Nauk. 253 (1980) 495–497].     

Chemical-Enzymatic Synthesis of 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of Natural Heterocyclic Bases and Their 5′-Monophosphates

Abstract

Treatment of O², 3′-anhydro-5′-O-trityl derivatives of thymidine (1) and 2′-deoxyuridine (2) with lithium azide in dimethylformamide at 150 °C resulted in the formation of the corresponding isomeric 3′-azido-2′, 3′-dideoxy-5′-O-trityl-β-D-ribofuranosyl N¹- (the major products) and N³-nucleosides (3/4 and 5/6). 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of thymidine [Thd(3′NH₂)], uridine [dUrd(3′NH₂)], and cytidine [dCyd(3′NH₂)] were synthesized from the corresponding 3′-azido derivatives. The Thd(3′NH₂) and dUrd(3′NH₂) were used as donors of carbohydrate moiety in the reaction of enzymatic transglycosylation of adenine and guanine to afford dAdo(3′NH₂) and dGuo(3′NH₂). The substrate activity of dN(3′NH₂) vs. nucleoside phosphotransferase of the whole cells of Erwinia herbicola was studied.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Eusiderins and other neolignans from an Aniba species

A previous report disclosed the presence of benzodioxan and bicyclo[3.2.1]octanoid neolignans in the benzene extract of the trunk wood of an Amazonian Aniba (Lauraceae) species. The chloroform extract of the same material contains additionally two new benzodioxan neolignans [rel-(7S,8R)-Δ^8′-7-hydroxy-3,4,5,5′-tetramethoxy-7.0.3′,8.0.4′-neolignan; rel-(7R,8R)-Δ^7′-3,4,5,5′-tetramethoxy-9′-oxo-7.0.3′,8.0.4′-neolignan], two new bicyclo[3.2.1]-octanoid neolignans [(7R,8S,1′S,2′S,3′S,4′R)-Δ^8′-2′,4′-dihydroxy-3,3′-dimethoxy-4,5-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan; (7R,8S,1′R,2′S,3′S)-Δ^8′-2′-hydroxy-3,3′,5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-4′-oxo-7.3′,8.1′-neolignan] and a hydrobenzofuranoid neolignan [(7S,8R,1′S,5′S)-Δ^8′-3,3′,5′-tri-methoxy-4,5-methylenedioxy-1′,4′,5′,6′-tetrahydro-4′-oxo-7.0.2′,8.1-neolignan].     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

Dimeric hydroanthracenes from the unripe seeds of Cassia torosa

From the unripe seeds of Cassia torosa three new dimeric hydroanthracene derivatives were isolated along with stigmasterol, sitosterol, campesterol, physcion-9-anthrone, torosachyrsone and the phlegmacins A₂ and B₂. The structures of the new derivatives were established as physcion-10, 10′-bianthrone, anhydrophlegmacin B₂ [2-(6′-methoxy-3′-methyl-3′, 8′, 9′-trihydroxy-1′-oxo-1′, 2′, 3′, 4′-tetrahydroanthracene-10′-yl)-1, 8-dihydroxy-3-methoxy-6-methyl-9-oxo-9, 10-dihydroanthracene] and torosanin [2-(6′-methoxy-3′-methyl-3′, 8′,9′-trihydroxy-1′-oxo-1′, 2′, 3′,4′-tetrahydroanthracene-5′-yl)-1, 8-dihydroxy-3-methoxy-6-methyl-9-oxo-9, 10-dihydroanthracene], respectively.     

Cinnamate and shikimate incorporation into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins: Are there alternative pathways?

The incorporation of shikimate-[¹⁴C] and cinnamate-[¹⁴C] into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins was studied in isolated petals of Petunia hybrida. According to the dilution values, the incorporation of shikimate-[¹⁴C] was about 3–6 times better than that of cinnamate-[¹⁴C]. However a comparison of the incorporation of the 2 precursors on a relative basis showed no significant differences in the relative proportions of the specific activities of the 3′,4′-dihydroxysubstituted cyanidin-3-monoglucoside and the 3′,4′,5′-trihydroxysubstituted delphinidin-3-monoglucoside. This result and the [¹⁴C]-incorporation behaviour of the 3′-methoxy-4′-hydroxysubstituted peonidin-3-monoglucoside do not support the hypothesis that there are alternative pathways of flavonoid biosynthesis.     

Alternative pathways of xanthone biosynthesis in cell cultures of Hypericum androsaemum L.

The biosynthesis of xanthones was studied in cell cultures of Hypericum androsaemum L. We have detected a new benzophenone synthase, for which the preferred substrate is benzoyl-CoA, itself supplied by 3-hydroxybenzoate:coenzyme A ligase. The stepwise condensation of benzoyl-CoA with three molecules of malonyl-CoA, catalyzed by benzophenone synthase, yields 2,4,6-trihydroxybenzophenone. This intermediate is subsequently converted by benzophenone 3′-hydroxylase, a cytochrome P450 monooxygenase. These biosynthetic steps, leading to the formation of 2,3′,4,6-tetrahydroxybenzophenone, represent an alternative pathway to that recently proposed for cell cultures of Centaurium erythraea [Peters et al., Planta (1997) in press].     

2′,5′-Adenylate and Cordycepin Trimer Cores: Metabolic Stability and Evidence for Antimitogenesis without 5′-Rephosphorylation

Abstract

The effect of the 2′,5′-adenylate and cordycepin trimer cores on DNA and protein synthesis in human umbilical cord lymphocytes, lymphoblasts, peripheral blood lymphocytes and Epstein-Barr virus infected lymphocytes and their metabolism in tissue culture medium have been studied. [³²P]Adenylate and [³²P]- and [³H]cordycepin trimer cores were synthesized either enzymatically or chemically and added to cells in culture. The half-lives of the 2′,5′-A₃ core and 2′,5′-3′dA₃ core in tissue culture were 3 and 17 hr, respectively. Chromatographic analysis of the TCA-soluble extracts of the lymphocytes and lymphoblasts treated with 2′,5′-[³H]A₃ showed that 0.25% of the ³²P was identified as AMP, ADP, ATP and inorganic phosphate. By the more sensitive 2′,5′-p₃A₄[³²P]pCp radiobinding assay, 2′,5′-A₃ was detected in the TCA supernatants; however, there was no 5′-rephosphorylation. With the [³H]- and [³²P]cordycepin trimer core, 0.55% and 1.3% of the radioactivity was in the TCA soluble extracts. Although there was no 5′-rephosphorylation as determined by radiobinding assay, the intact cordycepin trimer core was detected by tlc, radiobinding assay, and HPLC.

Furthermore, in two experiments, the concentration of the cordycepin trimer core bound to or taken up by the lymphocytes was three-fold greater than the concentration in the medium. 2′,5′-A₃ and 2′,5′-3′dA₃ cores were both antimitogenic, but did not inhibit protein synthesis.     

Rapid Ammonia Gas Transport Accounts for Futile Transmembrane Cycling under NH3/NH4+ Toxicity in Plant Roots

Futile transmembrane NH₃/NH₄⁺ cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH₃/NH₄⁺ toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH₃ or NH₄⁺) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope ¹³N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH₃ species, rather than the NH₄⁺ ion. Influx of ¹³NH₃/¹³NH₄⁺, which exceeded 200 µmol g^–1 h^–1, was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH₃ transport. Influx followed Michaelis-Menten kinetics for NH₃ (but not NH₄⁺), as a function of external concentration (K_m = 152 µm, V_max = 205 µmol g^–1 h^–1). Efflux of ¹³NH₃/¹³NH₄⁺ responded with a nearly identical K_m. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH₃ is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.Ammonia/ammonium (NH₃/NH₄⁺) toxicity in higher plants has resulted in crop reduction and forest decline (Pearson and Stewart, 1993; Vitousek et al., 1997; Britto and Kronzucker, 2002), biodiversity loss (Stevens et al., 2004; Bobbink et al., 2010), and species extirpation (de Graaf et al., 1998; McClean et al., 2011). These major ecological and economic problems have been aggravated by an accelerated global nitrogen (N) cycle caused primarily by the industrialized production and use of N fertilizers (Galloway et al., 2008; Gruber and Galloway, 2008). With increasing global population and demands on agricultural production, there is no sign of this trend easing: anthropogenic N fixation has reached 210 teragrams year^–1, an approximately 12% increase from 2005 and an approximately 1,300% rise from 150 years ago (Galloway et al., 2008; Fowler et al., 2013).Although considerable knowledge of the causes and mechanisms of NH₃/NH₄⁺ toxicity has accrued in recent years, our understanding of the key processes remains rudimentary (Gerendas et al., 1997; Britto and Kronzucker, 2002). A major hypothesis is that of futile transmembrane NH₄⁺ cycling, which proposes a pathological inability of root cells to restrict the primary entry of NH₄⁺ at high external concentrations ([NH₄+]_ext); many downstream toxicological events are contingent upon this entry (Britto et al., 2001b). In this model, a rapid, thermodynamically passive influx of NH₄⁺ is coupled to an active efflux of NH₄⁺ that is nearly as rapid, constraining normal cellular function and energetics and resulting in plant growth decline and mortality. This phenomenon is thought to occur in NH₄⁺-sensitive species such as barley (Hordeum vulgare) and, to a lesser extent, in tolerant species such as rice (Oryza sativa), which can be susceptible at higher thresholds (Balkos et al., 2010; Chen et al., 2013).Most soils are typically acidic, especially when [NH₄⁺] is high (i.e. in the millimolar range; Van Breemen et al., 1982; Bobbink et al., 1998; Britto and Kronzucker, 2002), and given the pKa of 9.25 for the conjugate pair NH₃/NH₄⁺, [NH₃] is generally low (Izaurralde et al., 1990; Weise et al., 2013). Consequently, the fluxes of NH₃ have largely been considered negligible (Britto et al., 2001a; Britto and Kronzucker, 2002; Loqué and von Wirén, 2004), in contrast to NH₄⁺ fluxes, which are well characterized physiologically (Lee and Ayling, 1993; Wang et al., 1993a, 1993b; Kronzucker et al., 1996) and at the molecular level (Rawat et al., 1999; von Wirén et al., 2000; Ludewig et al., 2007), at least at lower concentrations. However, the transport of NH₃ across membranes has received new attention in the light of evidence that some members of the aquaporin (AQP) family of transporters, a diverse and ubiquitous class of major intrinsic proteins (Maurel et al., 2008; Hove and Bhave, 2011), can mediate NH₃ fluxes in single-cell systems (Jahn et al., 2004; Holm et al., 2005; Loqué et al., 2005; Saparov et al., 2007). However, a convincing demonstration that AQPs transport NH₃ in planta is currently lacking. Given the unusually high capacity of AQP-mediated fluxes relative to those of ion channels and other transporters (Kozono et al., 2002), it is possible that sizable NH₃ fluxes can be conducted through AQPs, even at very low external NH₃ concentration ([NH₃]_ext).Here, we have critically reexamined the hypothesis that futile cycling is composed of cationic NH₄⁺ fluxes across the plasmalemma, of which an active efflux mechanism accounts for energetic demands directly contributing to toxicity (Britto et al., 2001b). We present evidence for the following alternative scenario: 1) futile cycling consists mainly of the passive electroneutral flux of the conjugate base NH₃; 2) such fluxes rapidly span both major membrane systems in root cells (i.e. plasmalemma and tonoplast); 3) AQPs mediate such fluxes; and 4) a thermodynamic equilibrium of NH₃ is established throughout the cell, resulting in hyperaccumulation of NH₄⁺ in the acidic vacuole. This evidence comes primarily from positron emission tracing with the short-lived radioisotope ¹³N, used to characterize the component fluxes of futile cycling at the cellular level in the model species barley. We have coupled this with ⁴²K⁺ radiotracing, to provide comparison with a well-understood cationic flux, as well as electrophysiological, respiratory, pharmacological, and histochemical analyses.     

Xanthones from Tovomita pyrifolium

The wood of Tovomita pyrifolium (Guttiferae) contains the novel tovopyrifolins A [1,6-dihydroxy-7-methoxy-5-prenyl-6′,6′-dimethylpyrano (2′,3′:3,2)xanthone], B (1,5-dihydroxy-3,4-dimethoxyxanthone) and C (1,3,5-trihydroxy-2-methoxyxanthone) and also the known tovophyllins A and B [structure revised to 1,6-dihydroxy-5-prenyl-6′, 6′-dimethylpyrano(2′,3′:3,2)-6″,6″-dimethylpyrano(2″,3″:7,8)xanthone].     

Inhibition by ammonia of intracellular cAMP accumulation in dictyostelium discoideum: Its significance for the regulation of morphogenesis

Two compounds, ammonia (NH₃) and 3′5′ cyclic AMP (cAMP) act as specific morphogens in regulating the development of Dictyostelium discoideum [1–11]. A previous study [12] demonstrated that NH₃ at concentrations that affect the course of morphogenesis completely inhibits the extracellular release of cAMP by aggregation competent cells incubated in shaken suspension. The present study extends this finding in two respects:

• 1 Exposure of aggregation competent cells to NH₃ (supplied as ammonium carbonate) is followed within a few minutes by the complete disappearance of intracellular cAMP. Subsequent removal of NH₃ is followed by a rapid, complete restoration of the level. Neither the disappearance nor the reappearance is affected by the presence of cycloheximide, an inhibitor of protein synthesis.
• 2 In a mutant strain of D discoideum, greatly increased sensitivity to NH₃ as a regulator of morphogenesis is coupled with a correspondingly increased sensitivity to NH₃ as an inhibitor of cAMP accumulation.

These results are consistent with a recently proposed [13, 14] model of morphogenetic regulation that is based on the supposition that NH₃, by inhibiting cAMP production, restricts cAMP accumulation to specified constrained areas within the developing multicellular aggregate and thereby dictates the course of morphogenesis and cytodifferentiation.     

A simple radioimmunoassay for 3',5', cyclic adenosine monophosphate.

A radioimmunoassay for 3′,5′ cAMP has been developed in which [³H]3′,5′ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is 0.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotides. Samples are prepared by ethanol precipitation. Tissue levels of 3′,5′ cAMP are comparable to those reported by others.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

Partial purification of the protein system controlling the breakdown of trehalose in baker's yeast.

The inactive form of trehalase as well as its activating protein have been partially purified from resting cells of baker's yeast using (NH₄)₂SO₄ fractionation and subsequent DEAE- and CM-cellulose column chromatography. For its activation by cyclic 3′,5′-AMP the system appeared to be dependent on the presence of ATP and a divalent cation such as Mg²⁺, Mn²⁺ or Co²⁺. No sensitivity towards the pH was observed in the range 6.0 – 7.5. The amount of active trehalase formed was determined by the preincubation time and the concentration of the proteins involved. The activating protein partly lost its dependence on cyclic 3′,5′-AMP during purification. The results presented suggest that this protein may be a protein kinase and that activation of trehalase is associated with phosphorylation of the enzyme protein.     

Enzymatic Preparation of 32P-Labeled β-L-2′, 3′-dd-5′ ATP and Its Use as a High-Affinity,Conformation-Specific Ligand for Labeling Adenylyl Cyclases

Abstract

An enzymatic method was developed for the preparation of unlabeled and [β-³²P]-labeled β-L-2′,3′-dd-5′ATP from the monophosphate with near quantitative yields. β-L-2′,3′-dd-5′ATP was a competitive and potent inhibitor of adenylyl cyclases (IC₅ ～ 30 nM). Upon uvirradiation β-L-2′,3′-dd-[β-³²P]-5′ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2′,5′-dd-3′ATP, a post-transition state, noncompetitive inhibitor.     

Conformations of cyclic 3',5'-nucleotides. Effect of the base on the synanti conformer distribution

The furanose and the phosphate rings of cyclic 3′,5′-nucleotides are locked in the ₄T³ and chair conformations respectively. The only variable which shows major conformational flexibility in these molecules is the rotation about the glycosyl bond which describes the orientation of the base relative to the sugar-phosphate bicyclic system. The glycosyl torsion angle has been analyzed for cyclic nucleotides with different purine and pyrimidine bases by use of conformational energy calculations. The results indicate that all the pyrimidine bases, U, T and C show a very strong energetic preference for the anti range of conformations. The calculations predict that among cyclic 3′,5′-purine nucleotides cyclic GMP and cyclic IMP favor the syn conformation to the anti by 95:5 and 70:30 respectively, while cyclic AMP shows a preference for the anti conformation to syn by 70:30. Thus the purines show a greater probability for the syn conformation than the pyrimidines in cyclic 3′,5′-nucleotides.     

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. $\text{249}$, 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. $\text{8}$, 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m₃², 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.     

Catalytic cycloalumination in steroid chemistry II: Selective functionalization of 2′-methylidene-2′,3′-ethano-(5α)-cholestane

The catalytic cycloalumination of 2′-methylidene-2′,3′-ethano-(5α)-cholestane with Et₃Al catalyzed by Cp₂ZrCl₂ was performed for the first time to give spiro[2′,3′-ethano-(5α)-cholestane-2′,3″-aluminacyclopentane] in a ～75% yield and with high stereoselectivity (>98%). The obtained cyclic organoaluminum compound was transformed in situ into heterocyclic spiran derivatives of 2′,3′-ethano-(5α)-cholestane.     

Hydroxyl Radical-Mediated Oxidation of Serotonin: Potential Insights into the Neurotoxicity of Methamphetamine

Abstract: When incubated with a hydroxyl radical (HO^?)-generating system (ascorbic acid/Fe²⁺-EDTA/O₂/H₂O₂), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3′-bis(2-aminoethyl)-5-hydroxy-[3,7′-bi-1H-indole]-2,4′,5′-3H-trione (7). The latter slowly cyclizes to 3′-(2-aminoethyl)-1′,6′,7′,8′-tetrahydro-5-hydroxyspiro[3H-indole-3,9′-[9H]pyrrolo[2,3-f]quinoline]-2,4′,5′(1H)- trione (9). A minor amount of T-4,5-D dimerizes to give 7,7′-bi-(5-hydroxytryptamine-4-one) (7,7′-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO^?-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO^? formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO^?-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO^? formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO^? might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.