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BackgroundEndometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterus causing chronic inflammation, severe pain, and infertility. However, the innate immunity of gamma-delta (γδ) T lymphocytes in endometriosis has not been characterized. Women with endometriosis present numerous endocrine and immune dysfunctions and elevated risk for endometrial, ovarian, and breast cancers. The tyrosine kinase EphA2 is often overexpressed in cancer including endometrial carcinoma.MethodsWe analyzed Vδ1 and Vδ2 γδ T cells in peripheral blood and paired peritoneal fluid samples in endometriosis patients (n = 19) and compared the counts with that of age- and sex-matched healthy donors (n = 33) using flow cytometry. Vδ1 and Vδ2 T cells isolated from healthy donors were used against KLE, RL-95, and Ishikawa endometrial tumor cells in 4 h flow cytometric cytotoxicity assays. The EphA2 blocking studies were performed using antibody, small-molecule inhibitor ALW-II-41-27, and the CRISPR/Cas9.ResultsWe determined Vδ1 T cells substantially reduced in patients’ peripheral blood (p p ConclusionsWe determined variable levels of Vδ1 and Vδ2 γδ T cells in endometriosis patients. We observed inherent cytotoxic reactivity of γδ T-cell subsets against endometrial cell lines. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of endometrial tumor killing mediated by Vδ1 γδ T cells. These results suggest that EphA2 is involved in tumor cell lysis and contributes to susceptibility to Vδ1 γδ T cells cytotoxic reactivity.

The semirigid binuclear ruthenium complex Delta,Delta-[mu-(11,11'-bidppz)(phen)(4)Ru(2)](4+) has been shown to rearrange slowly from an initial groove-bound nonluminescent state to a final intercalated emissive state by threading one of its bulky Ru(phen)(2) moieties through the DNA base stack. When this complex binds to poly[d(A-T)(2)], a further increase in emission from the complex is observed after completion of the intercalation, assigned to reorganization of the intercalated complex. We here report a study of the threading process in poly[d(A-T)(2)], in which the minor groove binding dye DAPI is used as an energy transfer probe molecule to assess the distribution of ruthenium complex during and also after the actual threading phase. The emission from DAPI is found to change with the same rate as the emission from the ruthenium complex, and furthermore, DAPI does not disturb the binding kinetics of the latter, justifying it as a good probe of both the threading and the reorganization processes. We conclude from the change in the emission from both DAPI and the ruthenium complex with time that DAPI-ruthenium interactions are most pronounced during the process of threading of the complex, suggesting that the complexes are initially threaded slightly anticooperatively and thereafter redistribute along the DNA to reach their thermodynamically most favorable distribution. The final distribution is characterized by a small but significant binding cooperativity, probably as a result of hydrophobic interactions between the complex ions despite their tetravalent positive charges. The mechanism of "shuffling" the complex along the DNA chain is discussed, i.e., whether the ruthenium complex remains threaded (requiring sequential base-pair openings) or if unthreading followed by lateral diffusion within the ionic atmosphere of the DNA and rethreading occurs.

Epoxidation and cis-hydroxylation of C-phenyl-delta(2,3)-glycopyranosides have been carried out with a view to developing C-aryl glycoside synthesis. Epoxidation of (2,3- and (6-O-tert-butyldimethylsilyl-2,3-dideoxy-D-erythro-hex-2-enopyranosyl)benzenedideoxy-D-erythro-hex-2-enopyranosyl)benzene gave predominantly the allo-adducts whatever the configuration at the anomeric center. Epoxidation of (4,6-di-O-tert-butyl-dimethylsilyl-2,3-dideoxy-D-erythro-hex-2-enopyranosyl)benzene gave the manno- and allo-adducts in a 89:11 and 40:60 ratios for the alpha- and beta-anomers, respectively. Hydroxylation of alpha-C-phenyl-(2,3)-glycopyranosides using OsO4 afforded the manno-adduct only, whatever the substituents at positions 4 and 6, whereas hydroxylation of (2,3-dideoxy-beta-D-erythro-hex-2-enopyranosyl)benzene and (4,6-di-O-tert-butyldimethylsilyl-2,3-dideoxy- beta-D-erythro-hex-2-enopyranosyl)benzene gave the manno- and allo-adducts in 25:75 and 80:20 ratios, respectively.

We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.

We investigated the applicability of Raman microprobe spectroscopy for determining the density of CO2 in fluid inclusions in minerals of mantle-derived xenolith samples. A separation (delta) between two Raman bands of CO2 due to Fermi resonance can be a reliable densimeter for CO2 fluid. The relationship between the density of CO2 (g/cm3) and delta (cm-1) can be expressed as: d = -0.03238697 delta 3 + 10.08428 delta 2 - 1046.189 delta + 36163.67. This equation was obtained from the Raman data on CO2 fluid with densities from 0.1 to 1.21 g/cm3, including super critical fluids at 58-59 degrees C. The delta value was constant with increasing temperature from room temperature to 200 degrees C. This indicates that the Raman densimeter is not affected by a possible rise in temperature, an artifact induced by the high flux of the incident laser. The minimum size of measurable inclusions is 1 micron, and the precision in the determination of delta is 0.1 cm-1, corresponding to 0.02 g/cm3 for inclusions of 1 micron in size. The precision can be better for larger inclusions. The micro-Raman densimeter can determine the density of CO2 fluid inclusions over a wide range. In particular, densities of gas and mixtures of gas and liquid phases, which cannot be measured by microthermometry, can be determined.

Kerr-gated picosecond time-resolved resonance Raman spectra of an enantiomerically pure ruthenium(II) diimine intercalator complex in water and acetonitrile are reported, affording the first direct temporally resolved evidence that its excited state is structurally different in the two solvents. Copyright © 2000 John Wiley & Sons, Ltd.

Treatment of p-tert-butylphenyl 4,6-di-O-benzyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside (1aα) or the 4,6-di-O-(tert-butyldimethylsilyl) analogue (1bα) with various functionalized arylmagnesium bromides in the presence of a catalytic amount of PdCl2(dppf) at 25 °C in THF afforded the corresponding unsaturated C-arylglycosides 2−14 having the α-configuration in quite good yields. Benzyl-, allyl-, and vinylmagnesium bromides gave also the corresponding unsaturated α-C-glycosides 15−18, although in lower yields. When the same reaction was performed in the presence of NiCl2(dppe) as the catalyst at −40 °C, only the formation of the corresponding unsaturated C-arylglycosides having the β-configuration was observed. As expected, reaction of phenylmagnesium bromide with p-tert-butylphenyl 4,6-di-O-benzyl-2,3-dideoxy-β-d-erythro-hex-2-enopyranoside (1aβ) in the presence of NiCl2(dppe) gave only the unsaturated β-C-phenylglycoside 2aβ, while palladium-catalyzed reaction led to the preponderant formation of C-ph...

Yu, W.-Z. and R. J. Bodnar. Interactions between Angiotensin Ii and delta opioid receptor subtype agonists upon water intake in rats. Peptides 18(2) 241–245, 1997.—Delta opioid receptor agonists, like those of mu and kappa receptors, stimulate water intake. To assess the relative contributions of delta 1 and delta 2 receptors in the modulation of water intake stimulated by Angiotensin II (AII), the present study examined the respective actions of [D-Pen 2 , D-Pen 5 ]-enkephalin (DPDPE: 5–20 ug, icv) and [D-Ala 2 , Glu 4 ]-Deltorphin (Delt II: 5–20 ug, icv) upon water intake per se, and upon AII (0.02–20 ng, icv)-induced hyperdipsia in rats. Both DPDPE and Delt II dose-dependently stimulated spontaneous water intake. An ineffective (5 ug) dose of DPDPE differentially altered water intake when paired with AII, significantly increasing intake at the 0.02 ng dose, not changing intake at the 0.2 ng dose and significantly decreasing intake at the 2 ng dose. In contrast, pairing ineffective doses of Delt II (5 ug) and AII (0.2 ng) significantly increased water intake that persisted when either the Delt II dose (1 ug) or the AII dose (0.02 ng) was lowered. However, neither delta 1 nor delta 2 opioid agonists significantly altered the ED 50 for AII-induced drinking. Thus, delta 2 opioid agonism appeared more consistent and reliable than delta 1 opioid agonism in stimulating water intake when paired with AII, and these data indicate interactions between delta 1 and delta 2 agonists and AII in mediating water intake.

The blood clam Barbatia lima subsp. from Amami-Oshima, Japan, expresses three types of haemoglobins in erythrocytes: a tetramer (alpha 2 beta 2), a homodimer (delta 2) and a polymer consisting of two kinds of chains, a 34 kDa two-domain (2D) globin and a delta chain. This is in sharp contrast to the congeneric clams B. reeveana (a North American species) and B. lima from Kochi, Japan, each containing a tetramer and a polymer consisting of the 2D globin, but not the delta chain. We have determined the cDNA-derived amino acid sequences of all four chains, alpha (163 residues), beta (155 residues), delta (152 residues) and 2D (308 residues) of B. lima (Amami-Oshima). The alpha chain has an extremely long N-terminal extension of 20 residues that may form a 'pre-A helix', and this makes the alpha chain the longest known globin. B. lima alpha and beta chains show about 50% sequence identity with the alpha and beta chains, respectively, of tetrameric haemoglobin from a related clam, Anadara trapezia. The B. lima homodimeric delta chain shows 71-74% identity with each of the two domains of the 2D chain, but only 39% identity with the homodimeric gamma chain of Anadara. In addition, the alignment of amino acid sequences of the delta chain and the two domains of the 2D chain revealed that the delta chain lacks one amino acid (Lys) at the C-terminus, suggesting that the C-terminal Lys (codon AAA or AAG) of the two domains of 2D chain could result from the stop codon TAA in the delta chain by nucleotide substitutions. These results, together with the fact that the delta and 2D chains form a polymeric haemoglobin, indicates that the delta chain is the ancestral single-domain globin for the 2D globin. The delta chain is expressed only in B. lima (Amami-Oshima), and appears to be a relic of molecular evolution.

Histidine usually exists in three different forms (including biprotonated species, neutral tau and pi tautomers) at physiological pH in biological systems. The different protonation and tautomerization states of histidine can be characteristically determined by C-13 and N-15 chemical shifts of imidazole ring. In this work, solid-state NMR techniques were developed for spectral editing of C-13 and N-15 sites in histidine imidazole ring, which provides a benchmark to distinguish the existing forms of histidine. The selections of C-13(gamma), C-13(delta 2), N-15(delta 1), and N-15(epsilon 2) sites were successfully achieved based on one-bond homo- and hetero-nuclear dipole interactions. Moreover, it was demonstrated that H-1, C-13, and 15 chemical shifts were roughly linearly correlated with the corresponding atomic charge in histidine imidazole ring by theoretical calculations. Accordingly, the H-1, C-13 and N-15 chemical shifts variation in different protonation and tautomerization states could be ascribed to the atomic charge change due to proton transfer in biological process. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.

1H-NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9-15-ppm region. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. delta-D-gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed. Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the C epsilon 1 and C delta 2 protons of an uncharged imidazole ring. We identify D as arising from the N epsilon 2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two-dimensional NOE spectra have provided additional information. Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered. The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7-10.6-ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.

Recently, we and others have shown the occurrence of TCR delta gene rearrangements in acute myeloid leukaemia (AML). In this study we describe the molecular characteristics of these rearrangements by the polymerase chain reaction (PCR) and the direct sequencing of PCR products. 11 rearrangements were characterized in blast cell samples from six patients. We found a heterogenous pattern of TCR delta gene rearrangements with involvement of V delta 1-5 regions. These findings differ from observations in T-ALL and B-cell precursor ALL, where predominantly usage of V delta 1 and V delta 2 regions has been described. Furthermore, extensive diversity of junctional sites was observed, including addition of up to 37 N nucleotides, nucleotide deletions at junction sites of V delta and J delta segments and usage of up to three D delta segments. The D delta 3 fragment was the most frequently used diversity element and was found in 10 rearrangements. Nine of the 11 rearrangements were non-functional, either incomplete or out of the reading frame. Therefore a functional TCR delta cannot be expressed in these myeloid blast cells.

Mitochondrial delta 3,5, delta 2,4-dienoyl-CoA isomerase, which catalyzes the conversion of 3,5-octadienoyl-CoA to 2,4-octadienoyl-CoA, was purified from rat liver 370-fold at almost 30% yield by a six-step purification procedure. The final preparation appeared to be homogeneous as judged by gel electrophoresis. The molecular weights of the native enzyme and its subunit(s) were estimated to be 126,000 and 32,000, respectively. The purification of delta 3,5, delta 2,4-dienoyl-CoA isomerase completes the characterization of the enzymes functioning in the NADPH-dependent pathway for the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms. This novel pathway may not be operative in peroxisomes because delta 3,5, delta 2,4-dienoyl-CoA isomerase was only detected in mitochondria. Substrates of this pathway are 2,5-dienoyl-CoAs formed from 5-enoyl-CoAs by acyl-CoA dehydrogenase. Two sequential isomerization reactions catalyzed by delta 3, delta 2-enoyl-CoAs isomerase and delta 3,5, delta 2,4-dienoyl-CoA isomerase, respectively, convert 2,5-dienoyl-CoAs to 2,4-dienoyl-CoAs, which are reduced by NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) before reentering the beta-oxidation spiral.

The dependence of the optical and spin-Hamiltonian parameters of NiF65- and NiF43- D4h units, with a b1g* ( approximately x2-y2) unpaired electron, upon the equatorial (Req) and axial (Rax) Ni+-F- distances has been studied through multiple-scattering X alpha and self-consistent charge extended Huckel methods. Both methods lead to the following main conclusions: (i) A charge-transfer transition like eu( pi + sigma ,eq) to b1g*( approximately x2-y2) (termed Eu) is more sensitive than a crystal-field one like b2g* ( approximately xy) to b1g*( Delta 1) or eg* to b1g*( Delta 2) to variations in Req. The change experienced by Eu mainly reflects that of Vel(M)-Vel(L), where Vel(M) and Vel(L) are the electrostatic potentials experienced by an electron placed on metal and ligands, respectively, (ii) As regards the unpaired spin densities onto nLp and nLs ligand valence orbitals (termed fsigma and fs respectively), it is found that fsigma >>fs but fs is much more sensitive than fsigma to changes in Req. The microscopic origin of this relevant fact is explained in detail. (iii) The removal of axial ligands, keeping Req constant, induces a decrease of Eu as a result of the diminution of the electrostatic repulsion, Vel (M), and produces a slight increase of Delta 1 and a more important one of Delta 2. (iv) The dependence of g//-g0 and gperpendicular to -g0 on Req essentially reflects that of Delta 1-1 and Delta 2-1, respectively, because of the low covalency (fsigma approximately=2%).

NMR studies on δ-decanolide samples obtained from 1 and 5 in reduction experiments with baker's yeast in D 2 O and H 2 O, respectively, show that the double bond saturation involves beta re-face trans formal addition of hydrogen atoms, arising to a large extent from water. The reduction does not depend on the absolute configuration at position 5 and shows a kinetic preference for the ( R ) enantiomer.

Values of ( delta beta / delta P)T derived thermodynamically from the zero pressure ultrasonic data of Niksch and co-workers (1980) indicate that the absolute magnitude of the volumetric thermal expansion coefficient beta should be initially enhanced by pressure both at very low temperatures, where beta is negative, and at higher temperatures when beta becomes positive. Values of ( delta 2(CP/T)/ delta P2)T, similarly derived, are in qualitative agreement with measurements of CP under pressure above 0.5 K, but at lower temperatures exacerbate an apparent inconsistency with thermal expansion data noted earlier by Brodale and co-workers (1986); it is unlikely that this anomaly is due solely to sample variation.

Photodegradation of 4''-(epimethylamino)-4''-deoxyavermectin B 1a as thin films on glass, using artificial light, resulted in the formation of multiple photodegradates. Six primary degradates and two secondary degradates were identified. The primary degradates formed were 8,9-Z-4''-(epimethylamino)-4''-deoxyavermectin B 1a (a geometric isomer), delta 2,3-4''-(epimethylamino)-4''-deoxyavermectin B 1a (a positional isomer)(.)

[formula: see text] An efficient strategy for the conversion of 1,2-diketones into saturated 1,6-diketones and delta 2,3/delta 3,4-unsaturated congeners thereof is reported.

Upon disulfide bond reduction, the alpha 2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.

Naive and primed alpha beta T cells can be distinguished on the basis of their differential expression of CD45RA and CD45RO, respectively. The present study indicates that these CD45-isoforms also identify naive and primed maturational stages of gamma delta T cells and natural killer (NK) cells. In peripheral blood, all V gamma 9-V delta 2 gamma delta T cells reportedly express CD45RO whereas all V delta gamma delta T cells lack CD45RO. Here, we show that these CD45RO- V delta gamma delta T cells all express CD45RA and the CD45RO+ V.9-V delta 2 gamma delta cells lack expression of CD45RA. The V delta T cells acquired CD45RO expression and lost part of their surface CD45RA, following in vitro activation with phytohaemagglutinin or IL-2. Also the CD3-CD16+ NK cells in peripheral blood that are uniformly CD45RA+ CD45RO- completely converted to the CD45RA-CD45RO+ phenotype upon in vitro activation. Moreover, all cloned V.9-V delta 2 and V delta 1 T cells and NK cells express CD45RO and lack expression of CD45RA. Our results strongly suggest that CD45RA and CD45RO are genuine markers for naive and primed lymphocytes that represent distinct differentiation lineages.

We have now found for the first time that nickel peroxide (NiO 2 ) functions as a superior oxidizing agent for the triazoline ring system in general. Dehydrogenation occurs exclusively and no side products are observed. NiO 2 oxidation of 1,5-substituted 1,2,3-triazolines thus provides an efficient general route for the synthesis of a wide variety of both, 1,5-diaryl- and 1-aryl-5-heteroaryl-1H-1,2,3-triazoles

The low-frequency FeCN vibrations of cyanoferric myeloperoxidase (MPO) and horseradish peroxidase (HRP) have been measured by resonance Raman spectroscopy. The ordering of the frequencies of the predominantly FeC stretching and FeCN bending normal vibrational modes in the two peroxidases differs. These normal mode vibrations are identified by their wavenumber shifts upon isotopic substitution of the cyanide ligand. For MPO, the stretching mode nu 1 (361 cm-1) occurs at a lower frequency than the bending mode delta 2 (454 cm-1). For HRP, the order is reversed as nu 1 (456 cm-1) is at a higher frequency than delta 2 (404 cm-1). Normal coordinate analyses and model complexes have been used to address the origin of this behavior. The nu 1 stretching frequencies in cyanide complexes of iron porphyrin and iron chlorin model compounds are similar to one another and to that of HRP. Thus, the inverted order and altered frequencies of the nu 1 and delta 2 vibrations in MPO, relative to those in HRP and the model compounds, are not inherent to the proposed iron chlorin prosthetic group in MPO but, rather, are attributed to distinct distal environmental effects in the MPO active site. The normal coordinate analyses for MPO and HRP showed that the nu 1 and delta 2 vibrational frequencies are not pure; the potential energy distributions for these modes respond not only to the geometry but also to the force constants of the nu(FeC) and delta(FeCN) internal coordinates.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray diffraction analysis (at 2.1-A resolution) of an acid alpha-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca2+ with an unusually high number of eight ligands (O delta 1 and O delta 2 of Asp175, O delta of Asn121, main-chain carbonyl oxygens of Glu162 and Glu210, and three water molecules). A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) alpha-amylase was also refined in a new crystal at 2.1-A resolution. The structure of this homologous (over 80%) enzyme and additional kinetic studies support all the structural conclusions regarding both calcium-binding sites.

Studies of the perturbing effect of chiral solvating agents (CSAs) namely the fluoroalcohols5aand5bupon the NMR spectra of chiral Δ2-thiazolines1presenting interesting insecticidal properties demonstrated the ability of these CSAs to afford diastereomeric solvates from these substrates providing their enantiomeric discrimination. Thus, for five of the six tested Δ2-thiazolines1Aand1Bthere is at least one possibility to proceed to their enantiomeric discrimination either by1H or19F NMR using mostly5bas CSA.

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.

International audience; Studies of the perturbing effect of chiral solvating agents (CSAs) 5a and mostly of 5c upon the NMR spectra of chiral Delta(2)-oxazoline 1 demonstrated the ability of these fluoroalcohols to afford diastereomeric solvates from these solutes. Thus, for all tested Delta(2)-oxazolines 1Aa-d, 1Ba, and 1e there is at least one possibility to proceed to their enantiomeric discrimination either by (1)H or (19)F NMR using these CSAs (see Fig. 1). NMR results are discussed from substrate and CSA structure standpoints and a solvation model is proposed on the basis of the inequivalence senses generally observed. Then the method was applied to extracts of incubated locust tissues obtained by solid phase extraction (SPE) after a partial unmasking of the substrate 1.

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.

A series of delta 2-isoxazoline derivatives structurally related to Broxaterol 1 and Falintolol 3 has been prepared and evaluated for their binding affinity to beta 1- and beta 2-adrenergic receptors. Among the tested compounds only the 3-isopropenyl anti derivative 4d is as active as the reference compounds. An electron-releasing group, probably operating through a pi-pi interaction, in the 3-position of the isoxazoline nucleus greatly enhances the affinity of the compounds. Conversely, the closest analogs of Broxaterol (3-bromo delta 2-isoxazolines 4a and 5a) are at least one order of magnitude less active than the model compound 1. Throughout the series of derivatives the anti stereoisomers are invariably more active than their syn counterparts.

1. The role of the glutamate receptor subunit delta 2 in the induction of cerebellar long-term depression (LTD) was investigated by application of antisense oligonucleotides. The delta 2 subunit is selectively localized to Purkinje cells (PCs), with the highest levels being in the PC dendritic spines, where parallel fibers are received and where cerebellar LTD is expressed. 2. Immunocytochemical analysis of calbindin-positive PCs revealed that both the dendritic and somatic expression of delta 2 was reduced in antisense-but not in sense-treated cultures. An antisense oligonucleotide directed against the related subunit delta 1 did not affect the expression of delta 2 in PCs. 3. Cerebellar LTD may be reliably induced in a preparation of cultured embryonic cerebellar neurons from the mouse when parallel and climbing fiber stimulation are replaced by brief glutamate pulses and strong, direct depolarization of the PC, respectively. Application of an antisense oligonucleotide directed against delta 2 completely blocked the induction of LTD produced by glutamate/ depolarization conjunctive stimulation. A delta 2 sense oligonucleotide or an antisense oligonucleotide directed against the related delta 1 subunit had no effect. 4. The effect of the delta 2 antisense oligonucleotide was not related to attenuation of calcium influx via voltage-gated channels or calcium mobilization via metabotropic glutamate receptors, as assessed with fura-2 microfluorimetry. Current flow through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-associated ion channels also appeared unaltered. All three of these processes have previously been shown to be required for cerebellar LTD induction. The observation that delta 2 is involved in a metabotropic-glutamate-receptor-independent signaling pathway that is required for LTD induction supports the view that delta 2 participates in the formation of a novel postsynaptic receptor complex.

Calbindin D28K binds 3 mol of terbium per mol of protein. To determine which of six EF-hand structures in the protein are responsible for terbium binding, we constructed three mutant forms of this protein, one lacking EF-hand 2 (RCaBP delta 2), the other lacking EF-hands 2 and 6 (RCaBP delta 2,6), and the third containing only EF-hands 3 and 4 (RCaBP delta 1,2,5,6), and examined their binding properties by fluorescence techniques. Full-length calbindin D28K and RCaBP delta 2 and RCaBP delta 2,6 bound 3 mol of terbium per mol of protein with high affinity. Thus, EF-hand domains 2 and 6 are not essential for calcium binding to the proteins, and an absence of EF-hands 2 and/or 6 does not alter the pattern of terbium binding to the protein. Using resonance energy transfer from tryptophan residues, one of the high affinity terbium-binding sites (site A) had a greater affinity than the other two sites (sites B and C) of each protein. Site A was filled before the other two sites. Calcium competition experiments showed that a greater amount of calcium was required to displace terbium from site A than from sites B or C. Energy transfer experiments from terbium to holmium showed that two of the terbium-binding sites are in close proximity while the third site is distant from the other two sites. To determine whether EF-hand 3 or 4 was responsible for binding of terbium, we examined the terbium binding properties of a delta 1,2,5,6 RCaBP construct. The truncated protein RCaBP delta 1,2,5,6 contained a single terbium-binding site. Analysis of the terbium binding to RCaBP delta 1,2,5,6 construct showed that site 4 bound terbium, whereas site 3 did not. Analysis of the terbium binding characteristics of the proteins suggests that EF-hands 1, 4, and 5 of rat brain calbindin D28K are responsible for terbium binding.

A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P P-E2(Na3) P-E2(Na2) P-E2(Na) P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.

The N-benzyl group of N-benzylnaltrindole (1, BNTI), a potent and selective delta 2 opioid receptor antagonist, was employed as a scaffold to hold electrophilic moieties (isothiocyanate and haloacetamide) in an effort to obtain selective affinity labels (2-4 and 8-11). The corresponding acetamide derivatives (5-7) also were synthesized to serve as nonelectrophilic controls. The o- and p-isothiocyanates (2 and 4) and the haloamides (8-11) were selective delta opioid receptor antagonists in the mouse vas deferens (MVD) preparations, while the meta isomer 3 was a delta-selective full agonist (IC50 = 5 nM). The fact that the effect of 2 and 4 was found to increase as a function of time in MVD suggests a covalent mechanism for the wash resistant component. The m-isothiocyanate 3 was found to be a delta-selective and irreversible agonist in the MVD, and it is suggested that it may be covalently binding to an agonist recognition site. In the mouse abdominal stretch antinociceptive assay, compounds 2-4 and 9 were delta-selective antagonists but exhibited delta 2/delta 1 selectivity ratios than that of BNTI.

Gamma/delta T cells may act as a first line of defence and respond to stress signals from the surrounding tissue. In the present investigation the occurrence of gamma/delta T cells was studied in the human skin after application of heavy metal salts by a routine epicutaneous patch-testing procedure. Gamma/delta cells were not found in normal skin. They were observed in all 14 allergic or irritant patch-test reactions to gold chloride and in 6/8 such reactions to mercuric chloride, where they comprised 16 +/- 6% and 15 +/- 6%, respectively, of the CD3+ cells in the dermis. They were also epidermotropic. Very few of these cells were found in reactions to salts of nickel and silver, except that they were increased in hair follicle epithelium in a reaction to silver nitrate. The gamma/delta cells expressed the V delta 2 and the V gamma 2 gene segments and were CD4-8-, indicating that they had the same phenotype as gamma/delta lymphocytes in the peripheral blood. Moreover, they were 'memory' T cells. These results indicate that gamma/delta lymphocytes play a role in the skin defence against highly reactive heavy metals.

The temperature dependence of the efficiency of oxidative refolding was examined for hen lysozyme three-disulfide derivatives produced in Escherichia coli. Each derivative was designed to lack one of the four disulfide bridges in authentic lysozyme: delta 1 (Cys6-->Ser, Cys127-->Ser), delta 2 (Cys30-->Ser, Cys115-->Ser), delta 3 (Cys64-->Ser, Cys80-->Ser), delta 4 (Cys76-->Ser, Cys94-->Ser), delta 2Ala (Cys30-->Ala, Cys115-->Ala), and delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for delta 1 (19 degrees C) and highest for delta 4Ala (30 degrees C). The chromatographically purified, completely refolded three-disulfide species were not stable above the optimal refolding temperature in the presence of glutathione. The stability of each of them was determined from the far-UV CD thermal denaturation measurement at pH 3.9 in the absence of glutathione, where the denaturation was reversible. The transition temperature was lowest for delta 1 and highest for delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were found to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for delta 1 in detail. The optimal temperature for refolding increased by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability in the presence of glycerol of refolded and purified delta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Duck lens delta-crystallins were the translational products of two tandem arranged genes, delta 1 and delta 2. Two fractions of the delta-crystallin (delta a and delta b) were resolved when the duck eye ball lens extract was passed through a Pharmacia-LKB Q-Sepharose column. These fractions were further separated by chromatofocusing into multiple forms with pI value ranging from 5.05 to 5.45. The charge heterogeneity of delta-crystallins was also demonstrated on the polyacrylamide gel when electrophoresis was performed under nonreducing conditions. All of those multiple forms possessed endogenous argininosuccinate lyase activity with activation energy approximately 12.5 +/- 1.6 kcal/mol. delta b-Crystallins showed higher enzyme activity than delta a-crystallins. Slightly different kinetic parameters were observed for the multiple forms of delta b-crystallins. On the other hand, delta a-crystallins could be further divided into two subgroups according to their kinetic parameters. There is 12-fold difference in kcat value between these two subgroups. delta a-Crystallins also have lower Km values than the delta b-crystallins. When examined by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate, the multiple forms were shown to be composed of subunits with similar M(r) of 55,000. All of these forms showed same antigenicity toward the rabbit anti-duck delta-crystallin antiserum; however, different carbonyl contents were observed for these forms, indicating that the origin of these multiple forms was due to post-translational oxidative modification of the protein molecules. Two delta-crystallin forms were demonstrated to be the N-truncated delta 2-crystallin, lacking the first eight amino acid residues from N-terminus. Modification and/or truncation of the proteins resulted in changing of the intrinsic tryptophan and tyrosine fluorescence. Those forms with higher pI values were shown to be much more thermostable than those with lower pI values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.