[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.     

Distribution and characterization of the opioid octapeptide met5-enkephalin-arg6-gly7-leu8 in the gastrointestinal tract of the rat

Summary The distribution and characterization of the opioid octapeptide met⁵-enkephalin-arg⁶-gly⁷-leu⁸ (met⁵-enk-arg⁶-gly⁷-leu⁸) within the gastrointestinal tract of the rat has been determined by immunohistochemistry and radioimmunoassay by use of a newly developed antibody to met⁵-enk-arg⁶-gly⁷-leu⁸. With both techniques, met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactivity (met⁵-enk-arg⁶-gly⁷-leu⁸IR) was detected in all regions of the gastrointestinal (GI) tract except the esophagus. The highest concentration of immunoreactive met⁵-enk-arg⁶-gly⁷-leu⁸ was observed in the colon, while intermediate concentrations were found in the stomach, duodenum, jejunum, and ileum. Immunostained somata were observed chiefly in the myenteric plexus; immunostained processes were present primarily in the myenteric plexus and the circular muscle layer. This distribution pattern is similar to that previously observed with antiserum to met⁵-enkephalin-arg⁶-phe⁷ (met⁵-enk-arg⁶phe⁷). Chromatographic analysis of met⁵-enk-arg⁶-gly⁷leu⁸-immunoreactive peptides extracted from the GI tract revealed the presence of an immunoreactive peptide of high molecular weight which accounted for approximately three-quarters of met⁵-enk-arg⁶-gly⁷-leu⁸-IR in both stomach and colon. These findings suggest a role for peptides related to the octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ in the regulation of GI function.     

The Na+,K+,2Cl−-cotransport system in HeLa cells: Aspects of its physiological regulation

We have previously reported on the biochemical properties of a Na⁺,K⁺,2Cl^?-cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na⁺,K⁺,2Cl^?-cotransport in HeLa cells was studied by ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺/²²Na⁺ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on ⁸⁶Rb⁺ flux, mediated by the bumet-anide-sensitive Na⁺, K⁺, 2Cl^?-cotransport system and the ouabain-sensitive Na⁺/K⁺-pump, were investigated. ANP reduced bumetanide-sensitive ⁸⁶Rb⁺ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive ⁸⁶Rb⁺ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a β-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive ⁸⁶Rb⁺ influx. Furthermore, efflux of ⁸⁶Rb⁺ and ²²Na⁺ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na⁺, K⁺, 2Cl^?-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α-methyl-aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na⁺, K⁺, 2Cl^?-cotransport-mediated ⁸⁶Rb⁺ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na⁺/K⁺-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-cGMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na⁺/K⁺-pump.     

Increased intratumoral IL-22-producing CD4+ T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival

IL-22-producing CD4⁺ T cells (IL-22⁺CD4⁺ T cells) and Th22 cells (IL-22⁺IL-17^?IFN-γ^?CD4⁺ T cells) represent newly discovered T-cell subsets, but their nature, regulation, and clinical relevance in gastric cancer (GC) are presently unknown. In our study, the frequency of IL-22⁺CD4⁺ T cells in tumor tissues from 76 GC patients was significantly higher than that in tumor-draining lymph nodes, non-tumor, and peritumoral tissues. Most intratumoral IL-22⁺CD4⁺ T cells co-expressed IL-17 and IFN-γ and showed a memory phenotype. Locally enriched IL-22⁺CD4⁺ T cells positively correlated with increased CD14⁺ monocytes and IL-6 and IL-23 detection ex vivo, and in vitro IL-6 and IL-23 induced the polarization of IL-22⁺CD4⁺ T cells in a dose-dependent manner and the polarized IL-22⁺CD4⁺ T cells co-expressed of IL-17 and IFN-γ. Moreover, IL-22⁺CD4⁺ T-cell subsets (IL-22⁺IL-17⁺CD4⁺, IL-22⁺IL-17^?CD4⁺, IL-22⁺IFN-γ⁺CD4⁺, IL-22⁺IFN-γ^?CD4⁺, and IL-22⁺IL-17⁺IFN-γ⁺CD4⁺ T cells), and Th22 cells were also increased in tumors. Furthermore, higher intratumoral IL-22⁺CD4⁺ T-cell percentage and Th22-cell percentage were found in patients with tumor-node-metastasis stage advanced and predicted reduced overall survival. In conclusion, our data indicate that IL-22⁺CD4⁺ T cells and Th22 cells are likely important in establishing the tumor microenvironment for GC; increased intratumoral IL-22⁺CD4⁺ T cells and Th22 cells are associated with tumor progression and predict poorer patient survival, suggesting that tumor-infiltrating IL-22⁺CD4⁺ T cells and Th22 cells may be suitable therapeutic targets in patients with GC.     

Triple-Resonance Methods for Complete Resonance Assignment of Aromatic Protons and Directly Bound Heteronuclei in Histidine and Tryptophan Residues

A set of three experiments is described which correlate aromatic resonances of histidine and tryptophan residues with amide resonances in ¹³C/¹⁵N-labelled proteins. Provided that backbone ¹H and ¹⁵N positions of the sequentially following residues are known, this results in sequence-specific assignment of histidine ¹H^δ2/¹³C^δ2 and ¹H^ε1/¹³C^ε1 as well as tryptophan ¹H^δ1/¹³C^δ1, ¹H^ζ2/¹³C^{ζ 2}, ¹H^η2/¹³C^η2, ¹H^ε3/¹³C^ε3, ¹H^ζ3/¹³C^ζ3 and ¹H^ε1/¹⁵N^ε1 chemical shifts. In the reverse situation, these residues can be located in the ¹H–¹⁵N correlation map to faciliate backbone assignments. It may be chosen between selective versions for either of the two amino acid types or simultaneous detection of both with complete discrimination against phenylalanine or tyrosine residues in each case. The linkages between δ-proton/carbon and the remaining aromatic as well as backbone resonances do not rely on through-space interactions, which may be ambiguous, but exlusively employ one-bond scalar couplings for magnetization transfer instead. Knowledge of these aromatic chemical shifts is the prerequisite for the analysis of NOESY spectra, the study of protein–ligand interactions involving histidine and tryptophan residues and the monitoring of imidazole protonation states during pH titrations. The new methods are demonstrated with five different proteins with molecular weights ranging from 11 to 28 kDa.     

Inhibition of 86Rb+ and 22Na+ efflux of Ehrlich lettré tumor cells by Ca2+.

The mechanism of the protective effect of Ca²⁺ on cellular K⁺ content was studied by examination of the effect of Ca²⁺ on efflux of the K⁺ analog, ⁸⁶Rb⁺, from preloaded cells with the use of compounds which interfere with monovalent cation movements. Ca²⁺ decreased ⁸⁶Rb⁺ efflux to the same extent in the presence and absence of ouabain, suggesting that Ca²⁺ did not alter the activity of the (Na⁺ + K⁺)-adenosine triphosphatase pump. Ca²⁺ exerted a similar protective effect in the presence of furosemide, an inhibitor of K⁺-K⁺ exchange, indicative that Ca²⁺ was not inhibiting this pathway. Since Ca²⁺ did not influence these pathways, it is concluded that Ca²⁺ exerts its primary effect by slowing passive diffusion. In support of this, Ca²⁺ also slowed ²²Na⁺ efflux. In addition, ethanol-induced leakage of ⁸⁶Rb⁺ was reversed by extracellular Ca²⁺, suggestive of a Ca²⁺-membrane phospholipid interaction.     

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells

Na⁺-ATPase of high-K⁺ and low-K⁺ sheep red cells was examined with respect to the sidedness of Na⁺ and K⁺ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na⁺ in the medium, i.e., at the cytoplasmic surface, Na_cyt⁺, the activation curves show that high-K⁺ Na⁺-ATPase has a higher affinity for Na_cyt⁺ compared to low-K⁺. The apparent affinity for Na_cyt⁺ is also increased by increasing the ATP concentrations in high-K⁺ but not low-K⁺. With Na_cyt⁺ present, Na⁺-ATPase is stimulated by intravesicular Na⁺, i.e., Na⁺ at the originally external surface, Na_ext⁺, to a greater extent in low-K⁺ than high-K⁺. Intravesicular K⁺ (K_ext⁺) activates Na⁺-ATPase in high-K⁺ but not in low-K⁺ vesicles and extravesicular K⁺ (K_cyt⁺) inhibits low-K⁺ but not high-K⁺ Na⁺-ATPase. Thus, the genetic difference between high-K⁺ and low-K⁺ is expressed as differences in apparent affinities for both Na⁺ and K⁺ and these differences are evident at both cytoplasmic and external membrane surfaces.     

Distribution of 46Sc and 51Cr in tumor-bearing animals and the mechanism for accumulation in tumor and liver

Tumor uptake rates, concentrations in mitochondrial fraction (containing lysosome) of liver and tumors, avid accumulations in connective tissue (especially inflammatory tissue) and binding substances in these tissues for ⁴⁶Sc³⁺ and ⁵¹Cr³⁺ were essentially similar to those for ⁶⁷Ga³⁺, ¹¹¹In³⁺, ¹⁶⁹Yb³⁺, ¹⁶⁷Tm³⁺, ⁹⁵Zr⁴⁺ and ¹⁸¹Hf⁴⁺. However, the main binding substance of ⁴⁶Sc³⁺ and ⁵¹Cr³⁺ in tumor and liver was the acid mucopolysaccharide (as described concerning ⁹⁵Zr and ¹⁸¹Hf) whose molecular weight exceeded 40,000, although the main binding substance of ⁶⁷Ga³⁺, ¹¹¹In³⁺, ¹⁶⁹Yb³⁺ and ¹⁶⁷Tm³⁺ was the acid mucopolysaccharide with a molecular weight of about 10,000.     

Nmr investigation of CYCLO7,10[C7,X9,C10,Nle12] analogues of the α-factor from saccharomyces cerevisiae

The cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²], cyclo^7,10[Cys⁷,D -Ala⁹,Cys¹⁰,Nle¹²], and cyclo^7,10[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] analogues of the α-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80 : 20) and aqueous solution by nmr spectroscopy. In addition, the cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²] α-factor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH dδ/dT data indicate that the cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²] α-factor adopts a type II β-turn in DMSO/water and that the cyclo^7,10[Cys⁷,D -Ala⁹,Cys¹⁰,Nle¹²] - and cyclo^7,10-[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] α-factor analogues adopt type II and type I/III β-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²] α-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III β-turn. In contrast, the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²] α-factor appears to adopt predominately a type II β-turn in DMSO/water. Quantitative NOE measurements of the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²]-, cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²]-, and cyclo^7,10[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] α-factors in DMSO/water were used to derive three-dimensional structures of the cyclo^7,10[Cys⁷,Pro⁸,X⁹Cys¹⁰] portion of these analogues. © 1994 John Wiley & Sons, Inc.     

Pesticidal and Receptor Binding Properties of Bacillus thuringiensis Cry1Ab and Cry1Ac δ-Endotoxin Mutants to Pectinophora gossypiella and Helicoverpa zea

Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. An understanding of their mechanisms of action is commercially important. In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's (³⁶⁸RR³⁶⁹) of Cry1Ab and Cry1Ac toxins were replaced with Ala (³⁶⁸AR³⁶⁹, ³⁶⁸RA³⁶⁹, ³⁶⁸AA³⁶⁹), Glu (³⁶⁸EE³⁶⁹), Phe (³⁶⁸FF³⁶⁹), His (³⁶⁸HH³⁶⁹), and Lys (³⁶⁸KK³⁶⁹). The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed. All seven mutants of Cry1Ab, excluding ³⁶⁸AR³⁶⁹, produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae. The Cry1Ab mutants, ³⁶⁸AR³⁶⁹, ³⁶⁸AA³⁶⁹, ³⁶⁸FF³⁶⁹, and ³⁶⁸HH³⁶⁹, lost toxicity; ³⁶⁸EE³⁶⁹ had reduced toxicity; whereas the more conserved change ³⁶⁸KK³⁶⁹ retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1Ac, ³⁶⁸AA³⁶⁹ and ³⁶⁸FF³⁶⁹, abolished the toxicity. Double mutant ³⁶⁸KK³⁶⁹ of Cry1Ac retained its toxicity against P. gossypiella, whereas single mutants ³⁶⁸AR³⁶⁹, ³⁶⁸RA³⁶⁹, and ³⁶⁸HH³⁶⁹ retained only reduced toxicity. All the mutants of Cry1Ab lost their toxicity against H. zea except ³⁶⁸KK³⁶⁹. In Cry1Ac single mutants, ³⁶⁸AR³⁶⁹ and ³⁶⁸RA³⁶⁹, reduction in the toxicity was observed. A double mutant of Cry1Ac, ³⁶⁸KK³⁶⁹, also retained reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence about the insecticidal property and inhibition of I_sc across the transepithelial membrane of the insect midgut. Received: 5 June 2000 / Accepted: 5 July 2000     

Molecular modeling and design of regioselectively addressable functionalized templates with rigidified three‐dimensional structures

Extensive conformational analysis of a series of β‐alkyl substituted cyclopeptides—cyclo(Pro¹–Xaa²–Nle³–Ala⁴–Nle⁵–Pro⁶–Xaa⁷–Nle⁸–Ala⁹–Nle¹⁰) and cyclo[Pro¹–Xaa²–Nle³–(Cys⁴– Nle⁵–Pro⁶–Xaa⁷–Nle⁸–Cys⁹)–Nle¹⁰] as well as their corresponding unsubstituted core structures cyclo(Pro¹–Xaa²–Ala³–Ala⁴–Ala⁵–Pro⁶–Xaa⁷–Ala⁸–Ala⁹–Ala¹⁰) and cyclo(Pro¹–Xaa²–Ala³–Cys⁴– Ala⁵–Pro⁶–Xaa⁷–Ala⁸–Cys⁹–Ala¹⁰) has been performed employing both the ECEPP/2 and the MAB force fields (Xaa = Gly, L ‐Ala, D ‐Ala, Aib, and D ‐Pro). Results show that (a) possible three‐dimensional structures of the cyclo(Pro¹–Gly²–Lys³–Ala⁴–Lys⁵–Pro⁶–Gly⁷–Lys⁸–Ala⁹–Lys¹⁰) molecule are not limited to a single extended “rectangular” conformation with all Lys side chains oriented at the same side of the molecule; (b) conformational equilibrium in monocyclic analogues obtained by replacements of conformationally flexible Gly residues for L ‐Ala, D ‐Ala, Aib, or D ‐Pro is not significantly shifted towards the target “rectangular” conformational type; and (c) introduction of disulfide bridges between positions 4 and 9 is a very powerful way to stabilize the target conformations in the resulting bicyclic molecules. These findings form the basis for further design of rigidified regioselectively addressable functionalized templates with many application areas ranging from biostructural to diagnostic purposes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 361–372, 1999     

A comparison of regulatory effects of chloride on nitrate uptake, and of nitrate on chloride uptake into Pisum sativum seedlings

Net nitrate uptake, ³⁶ClO^?₃/NO^?₃ influx and ³⁶Cl^? influx into Pisum sativum L. cv. Feltham First seedlings have been examined following growth in culture medium containing different combinations of chloride and nitrate. When young (6 days old) seedlings, that had been grown in the absence of N were used, nitrate accumulation stimulated net nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.99) while chloride accumulation inhibited nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.65). When nitrate was provided during growth there was no effect of chloride pretreatment on net nitrate uptake and there was little effect of total [NO^?₃+ Cl^?]_i on ³⁶ClO^?₃/NO^?₃ influx (r²= 0.26). A direct effect of Cl^? on ³⁶ClO^?₃/NO^?₃ influx was only found when seedlings had been starved of N for more prolonged periods (14 days). When moderate chloride was supplied during growth, ³⁶Cl^? influx was insensitive to nitrate or chloride accumulated, but significantly correlated with log_e [NO^?₃+ Cl^?]_i (r²= 0.75). When trace amounts of Cl^? were supplied during growth ³⁶Cl^? influx was inhibited by (a) NO^?₃ in the external medium and (b) Cl^? pretreatment, but was insensitive to NO^?₃ pretreatment. The sensitivity of ³⁶Cl^? influx to external nitrate was not found following Cl^? pretreatment in the absence of nitrate. The possibility that there are two populations of chloride carriers which differ in their sensitivity to external nitrate is discussed. Tentative schematic models to account for the regulation of nitrate and chloride uptake are proposed in the context of current hypotheses for regulation of ion transport and control systems theory.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

Base-pair exchange kinetics of the imino and amino protons of the 3′-phenazinium tethered DNA-RNA duplex,r(5′GAUUGAA3′):d(5′TCAATC3′-Pzn), and their comparison with those of B-DNA duplex

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [^5′r(G¹A²U³U⁴G⁵A⁶A⁷)^3′]:^5′p[d(T⁸C⁹A¹⁰A¹¹T¹²C¹³)]^3′-Pzn] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C¹G²T³A⁴C⁵G⁶)₂ (II), d[p(^5′T¹G²T³T⁴T⁵G⁶ G⁷C⁸)^3′]:d[p(^5′C⁹C¹⁰A¹¹A¹²A¹³C¹⁴A¹⁵)^3′] (III), d(C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶)₂ (IV) and d(C¹G²C³G⁴C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶C¹⁷G¹⁸C¹⁹G²⁰)₂ (V). This comparative study shows that the life-times τ_o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of ∼ 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the τ_o of the larger duplexes (IV) and (V): the τ_o for the base pair of T¹¹ and T¹² residues in the 20-mer (duplex V) are 2.9 ± 2.3 ms and 23.2 ± 8.9 ms, respectively, while the corresponding τ_o in the 12-mer (duplex IV) are 2.8 ± 2.2 ms and 17.4 ± 5.4 ms. It has also been shown that the total energy of activation (E_a) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the E_a of the short B-DNA duplex (E_a ≈ 28–47 kcal/mol).     

Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass

Experiments with intact plants of Lolium perenne previously grown with ¹⁴NO₃⁻ revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched ¹⁵NO₃⁻. The exuded ¹⁴NO₃⁻ was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the ¹⁴NO₃⁻ was observed. Influx of ¹⁵NO₃⁻ was significantly greater than influx of ¹⁴NO₃⁻ from solutions of identical NO₃⁻ concentration. Transferring plants to ¹⁴NO₃⁻ solutions after a six-hour period in ¹⁵NO₃⁻ resulted in efflux of the latter. Presence of Mg²⁺, rather than Ca²⁺, in the ambient ¹⁵NO₃⁻ solution resulted in a decidedly increased rate of ¹⁴NO₃⁻ efflux and a slight but significant increase in ¹⁵NO₃⁻ influx. Accordingly, net NO₃⁻ influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO₃⁻ within unstirred layers from the passive pathway to the active uptake site.     

Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen

Glutamine is the first major organic product of assimilation of ¹³NH₄⁺ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of ¹³NO₃⁻ by tobacco cells cultured on nitrate. The percentage of organic ¹³N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. ¹³NO₃⁻, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of ¹³N in glutamine, and a concomitant increase in the percentage of ¹³N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NH₄⁺ into glutamine more extensively than it inhibits the incorporation of ¹³N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when ¹³NH₄⁺ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of ¹³NH₄⁺ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NO₃⁻ into glutamate more strongly than it inhibits the incorporation of ¹³N into glutamine, suggesting that the assimilation of ¹³NH₄⁺ derived from ¹³NO₃⁻ may be mediated solely by the glutamine synthetase-glutamate synthase pathway.     

Inhibition of Photohemolysis Induced by m-Chloroperbenzoic Acid by Metal Complexes with SOD-mimetic Activity

Red blood cells (RBCs) are probably the most common target through the damaging action of reactive oxygen species on the cells. The photohemolysis activity of m-chloroperbenzoic acid (CPBA) was concentration- and exposure time-dependent. Twenty minutes photo exposure time and 200?μm of CPBA concentration were optimum to study the effect of generated superoxide (O₂^-) and hydroxyl (^?OH) radicals on RBCs. RBCs lysis photosensitized by CPBA was investigated in the presence of [(VL²O)(VL²H²O)]Cl⁶, [MnL²O]²Cl⁴2H²O, [FeL²Cl²]Cl H²O, [CoL²Cl²]⁴H²O or [ZnL²Cl²]H²O respectively, where L is 2-methylaminopyridine, with SOD-mimetic activities with the aim of ascertaining their protective activity towards the photo induced cell damage. The decrease of photolytic activity caused by these complexes was concentration-dependent and the maximum percentage of protective activity was 75, 70, 68, 57 or 24% for [(VL²O)(VL²H²O)]Cl⁶, [MnL²O]²Cl⁴ 2H²O, [FeL²Cl²]Cl H²O, [CoL²Cl²]⁴H²O or [ZnL²Cl²]H²O complex respectively, against the cell irradiated without addition of metal complexes. The comparison between the decrease of photolytic activity caused by these complexes and their SOD-mimetic activity of these metal complexes showed an appreciable correlation.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.