In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.     

Stimulation of photosynthesis by 2% oxygen at low temperatures is restored by phosphate

The effect of phosphate feeding on the influence of low (2%) oxygen on photosynthetic carbon assimilation has been investigated in leaf discs of spinach (Spinacia oleracea L.) at 12°C. The following observations were made. First, after the transition from 20% O₂ to 2% O₂, the rate of CO₂ uptake was inhibited at CO₂ concentrations between about 250 and about 800 l CO₂·l^-1. Second, phosphate feeding stimulated the rate of CO₂ uptake in 20% O₂ at higher concentrations of CO₂ (500–900 l·l^-1). Third, phosphate feeding stimulated the rate of CO₂ uptake in 2% O₂ at all but the highest (900 l·l^-1) and lowest 74 (l·l^-1) concentrations of CO₂ employed. Phosphate thereby restored the stimulation of photosynthesis by 2% O₂ and it did so over a wide range of lower temperatures. Fourth, oscillatory behaviour, however generated, was dampened by phosphate feeding, even at very low concentrations of CO₂. Contents of leaf metabolites were measured during the transition to 2% O₂ in control and phosphate-fed leaf discs. During this period the ratio glycerate-3-phosphate/triose phosphate rose steeply, but fell again only in the phosphate-treated leaf discs. These data, taken together with measured ATP/ADP ratios, showed that assimilatory power, the ratio [ATP]·[NAD(P)H]/[ADP]·[Pi]·[NAD(P)], decreased when leaves were exposed to 2% O₂, but that this decrease was minimised by previous feeding of phosphate. The mechanism of phosphate limitation is discussed in the light of the results.Abbreviations C_i intercellular concentration of CO₂ - RuBP ribulose-1,5-bisphosphate     

NAD+-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD+ hydrolysis

We previously demonstrated inhibition of Na⁺-dependent ³²P_i transport in canine renal brush-border membranes in association with NAD⁺-induced ADP ribosylation of membrane protein(s) and postulated that NAD⁺ inhibits P_i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD⁺ resulted in release of P_i which was prevented by EDTA. It was proposed that NAD⁺-mediated inhibition of ³²P_i transport might occur through this mechanism. To determine whether NAD⁺ inhibited ³²P_i transport by a mechanism other than or in addition to release of P_i, we compared Na⁺-dependent ³²P_i counterflow in brush-border membrane equilibrated with P_i or with P_i generated from NAD⁺. Release of P_i from NAD⁺ incubated with brush-border membrane was confirmed. The increased uptake of ³²P_i which was demonstrated in brush-border membrane equilibrated with P_i was not measured when intravesicular P_i was generated from a concentration of NAD⁺ which effected ADP-ribosylation of brush border membranes (100 μM NAD⁺). In contrast, increased uptake of ³²P_i was demonstrated when intravesicular P_i was generated from 1 μM NAD⁺ which did not effect ADP ribosylation. Mg²⁺-dependent ADP ribosylation of brush-border membrane incubated with NAD⁺ was demonstrated which persisted during the time interval of ³²P_i uptake measurements. Our findings are compatible with the hypothesis that NAD⁺-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P_i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg²⁺ and decreased ADP ribosylation.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Divalent cations modify adsorption of 5′-AMP onto precipitated calcium phosphate: A model for cation modulation of adsorptive processes in primitive aqueous environments

The adsorption of 5′-AMP onto precipitated calcium phosphate (CaPi) requires the presence of soluble calcium and this dependence exhibits a Michaelian-like behavior. This result suggests that the formation of a complex between 5′-AMP and free Ca²⁺ (CaAMP) is a prelude to the adsorption of the nucleotide in the solid matrix. At concentrations one order of magnitude higher, Mn²⁺ and Mg²⁺ can substitute for soluble Ca²⁺ in the adsorption of 5′-AMP onto solid CaPi. However, when added simultaneously with 5′-AMP to a heterogeneous mixture that contains CaPi and soluble Ca²⁺, Mn²⁺ and Mg²⁺ inhibit the adsorption of 5′-AMP in a concentration-dependent manner. This suggests the formation of complexes that are much less effective for 5′-AMP adsorption than the CaAMP complex. On the other hand, Mn²⁺ and Mg²⁺ cannot promote desorption of the nucleotide attached to the precipitate in the presence of soluble Ca²⁺ if they are added after adsorption has attained equilibrium. Although desorption of 5′-AMP can be obtained by a sequential dilution of the soluble phase with buffer and no nucleotide in a process that obeys a Langmuir equation, the lack of effect of Mn²⁺ or Mg²⁺ when adsorption has attained its maximal value suggests strong interactions between the CaAMP complex and the solid matrix when adsorption equilibrium is reached. The divalent cations present in the matrix also participate with different selectivity in the attachment of the CaAMP complex, indicating that a cation-exchange mechanism could have acted in the modulation of adsorptive/desorptive processes involving biomonomers and phosphate surfaces in primitive aqueous environments. Received: 11 December 1995 / Accepted: 5 April 1996     

Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry

The 8-methoxycarbonyloctyl glycosides of GlcNAc, Gal1-4Glc, Fuc1-2Fuc1-3GalNac and Fuc1-2Gal1-3[Fuc1-4]GlcNac were converted to primary amines by reaction with neat ethylenediamine and then coupled to bovine serum albumin (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advantages of proceeding under very mild conditions, yielding controlled incorporation values and can reliably be used for the coupling of very small amounts (mg) of oligosaccharide.This paper is dedicated to Sen-itiroh Hakomori on the occasion of his 65th birthday     

Aluminium polyphosphate complexes in the mycorrhizal basidiomyceteLaccaria bicolor: A27Al-nuclear magnetic resonance study

The techniques of ²⁷Al- and ³¹P-nuclear magnetic resonance (NMR) spectroscopy were used to investigate the interactions of aluminium with intracellular ligands within the mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton (S238). The vegetative mycelium was grown on medium containing 0.5 mM AlCl₃ for 0.5 to 3 d. The ²⁷Al-NMR spectra showed that aluminium was rapidly taken up and accumulated into polyphosphate complexes in the vacuole. Comparison with Al-polyphosphate complexes obtained in vitro on model systems indicated that Al forms at least three mixed-solvation complexes with Pi and polyphosphates, that there is more than one complex present under any set of conditions, and that the equilibrium between these complexes shifts dramatically with Al concentration in the medium. The high phosphate concentrations in the growth medium favoured the accumulation of the Al-polyphosphate complexes. When mycelium containing Al-polyphosphate complexes was transferred to Al-free nutrient solution for 9 d, the Alpolyphosphate complexes were not remobilized. The sequestration of Al in the polyphosphate complexes could therefore make a significant contribution to the protection of mycorrhizal plants against aluminium toxicity.Abbreviations NMR nuclear magnetic resonance - PolyP polyphosphate(s) - PP₁ terminal phosphate of PolyP - PP₃ middle phosphate of PolyP We thank Prof. Daniel Canet (Laboratoire de Méthodologie RMN, University of Nancy I, Vandceuvre-lès-Nancy, France) for his constant encouragement and Christine Delaruelle for skilled technical assistance in growing the fungal cultures. This work was supported by a research grant from the Commission of the European Communities (STEP-CT90-0059, Role of Ectomycorrhiza in Stress Tolerance of Forest Trees) to F.M. and a travel grant from the Institut National de la Recherche Agronomique to I.K.; R.C. is a recipient of a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada.     

Deep-water stromatolites andFrutexites Maslov from the early and Middle Jurassic of S-Germany and Austria

Summary Despite extensive discussions during the last 20 years stromatolites are still used by many geologists as unequivocal indicators of very shallow-water conditions. We investigated four stratigraphic units from the Lower and Middle Jurassic of southern Germany (Posidonien-Schiefer, Amaltheen-Ton) and of the Northern Calcareous Alps (Adneter Kalk, Klauskalk), which were formerly interpreted as shallow marine sediments by some authors due to the occurrence of stromatolites. Our interpretations of the macro-, micro- and ultrafacies of these sediments are not compatible with shallow-water settings. We therefore propose a deep-marine, aphotic origin of these stromatolites. Former interpretations of the Posidonien-Schiefer as a shallow-water deposit are mainly based on the occurrence of stromatolites. We favour the model of a temporarily stagnant, deep, aphotic basin for these planktonrich sediments. Particles resembling ooids, but lying within mudstones cannot be taken as evidence for shallow agitated water. They either formed within the mud or are allochthonous. The deep-water setting of the red limestone of the Alpine Early and Middle Jurassic is indicated by a lack of platform-typical components like coated grains and phototrophic benthos and by shells of plankton and nekton forming a major part of the sediment. Stromatolites occur on the steep slope of a drowned Rhaetian reef with an estimated relief of 50–100 m and immediately below and within radiolarian limestones, deposited below the aragonite compensation depth (ACD). The aphotic stromatolites show some morphological differences to their shallow water counterparts. In all of our sections they occurred during intervals of reduced sedimentation. They form only thin horizons and probably grew very slowly. Mineralizations by Fe−Mn oxides and phosphate are very common. The presence of a microbial film is evident from binding of sedimentary particles, but the nature of the microbes is not known. Growth habits within the very distinct environments of red limestone and black shales show some common features, but also clear differences. The microproblematicumFrutexites Maslov is a very common component in deep-water stromatolites, but may also itself form small crusts or dendrolites. It occurs in two different forms. Opaque, slender forms with indistinct outlines probably grew within the weakly lithified sediment. Thicker, transparent forms with well defined outlines are found in cavities and probably also grew on the seafloor. Well preserved specimens display an internal fabric of radially arranged fibres of Fe−Mn oxides and calcite. It is suggested that calcite or aragonite were one original mineralogy ofFrutexites, which was later replaced by Fe−Mn oxides or phosphate. It is not certain whetherFrutexites is an organic, biomineralized structure or an inorganic mineralization, but the variable mineralogy and growth forms in different environments point to an organic origin. But even if organic, the occurrence in cryptic habitats and negative phototactic growth-directions make it clear thatFrutexites was not phototrophic.