Preparation, X-ray structure and solution behavior of [Ag(9-EtGH-N7)2]NO3·H2O (9-EtGH=9-ethylguanine)

The preparation and X-ray structure of [Ag(9-EtGH-N7)₂]NO₃·H₂O(9-EtGH=neutral 9-ethylguanine) is reported. The compound crystallizes in the triclinic system, space group P with a=7.063(6), b=7.153(3), c=11.306(10) Å, α=83.36(6), β=76.66(7), γ=81.44(6)°. The cation is centrosymmetric with Ag(I) coordinated via two N7 positions and Ag---N7 bond lengths of 2.11(1) Å. Applying ¹⁰⁹Ag NMR spectroscopy, complex formation constants for both the 1:1 complex (log β₁=0.6) and the title compound (log β₂=1.6) in Me₂SO have been determined.     

Mesure des déclins de l'intensité et de l'anisotropie de fluorescence du pérylène en présence de membranes d'érythrocyte de pigeon: Comparaison avec un milieu isotrope visqueux

Using synchrotron radiation as the excitation light, we studied the fluorescence parameters of perylene incubated with pigeon erythrocyte membranes and with an isotropic viscous medium, the Primol 342 oil.From 4 to 37°C, we observed a single lifetime of 4.5 ns in the oil and two with the membrane (τ₁ = 1−1.4 ns and τ₂ = 5.4−6.1 ns). The dependence upon temperature of the rotation correlation time of perylene () in the oil was characteristic of an isotropic medium, whereas the limiting value of anitropy (r ∞) was zero. With the membrane, γ ∞ decreased from 0.14 to 0.06 and from 2.9 to 0.5 ns, indicating a greater amplitude and frequency of molecular motions.The addition of chlorpromazine, indomethacine, tetracaine, n-octylamine, octanol or octanoic acid to the membrane decreased the τ₁ and τ₂ values. This would stem from the desorganization of the membrane induced by the drugs.     

η-N-succinimidato complexes of iron, molybdenum and tungsten as reversible inhibitors of papain

Recently we have found that the metallocarbonyl complexes (η⁵-C₅H₅)M(CO)_x(η¹-N-maleimidato) (M = Fe, Mo, W; x = 2 or 3) bearing a maleimide function were irreversible inhibitors of the enzyme papain. To get further insight into the binding mechanism of these compounds we synthesized the related complexes (η⁵-C₅H₅)M(CO)_x(η¹-N-succinimidato) (M = Fe, Mo, W; x = 2 or 3) that lacked the ethylenic bond responsible for alkylation of the cysteine 25 thiol group in the papain‘s catalytic pocket. We performed kinetic studies of the interaction of the synthesized complexes towards papain. We found that they act as reversible inhibitors of the enzyme with IC₅₀ values in the range 480–1700 μM. Docking experiments confirmed binding of these complexes to the enzyme’s catalytic pocket.     

Expression, purification and characterization of recombinant Z α1-Antitrypsin—The most common cause of α1-Antitrypsin deficiency

α₁-Antitrypsin (α₁AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. α₁AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe α₁AT deficiency are caused by the Z variant (E342K) of α₁AT. The presence of the Z mutation results in misfolding and polymerization of α₁AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z α₁AT production. This has created a major impediment to studying the effect of the Z mutation on α₁AT. Here we report our attempts to produce recombinant Z α₁AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z α₁AT. Cytosolic expression of the Z α₁AT gene in P. pastoris was successful. Monomeric and active recombinant Z α₁AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z α₁AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z α₁AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies.     

Cloning and Characterization of a P2X Receptor Expressed in the Central Nervous System of Lymnaea stagnalis

P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC₅₀ 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC₅₀ of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC₅₀ values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn²⁺ and Cu²⁺ ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.     

Extrahelical cytosine bases in DNA duplexes containing d[GCC]n·d[GCC]n repeats: detection by a mechlorethamine crosslinking reaction

The cytosine–cytosine (C–C) pair is one of the least stable DNA mismatch pairs. The bases of the C–C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an ‘extrahelical’ location. Here, using DNA duplexes with d[GCC]_n·d[GCC]_n fragments containing C–C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC]·d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C–C mismatch pairs C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆, we show that two mechlorethamine crosslinks form between C₁₃ and C₂₉ and between C₁₀ and C₃₂, in addition to crosslinks at C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆ (we have reported previously the crosslinking of formal C–C pairs by mechlorethamine). We interpret the formation of the C₁₃–C₂₉ and C₁₀–C₃₂ crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C–C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5′-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C₇–C₂₉ and C₁₀–C₂₆ crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC]_n·d[GCC]_n repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC]_n·d[GCC]_n repeat tracts.     

The inducible and cytochrome P450-containing dehydroepiandrosterone 7α-hydroxylating enzyme system of Fusarium moniliforme

7α-Hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7α-position, with minor hydroxylation occurring at the 7β-position. Constitutive 7α-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7α-hydroxylation via an increase in protein synthesis. DHEA 7α-hydroxylase was found to be mainly microsomal, and the best production yields of 7α-hydroxy-DHEA (28.5 ± 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (K_M=1.18 ± 0.035 μM and V_max=909 ± 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7α-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7α-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7α-hydroxylase; (iii) this enzyme complex contains a cytochrome P450.     

Paroxetine suppresses recombinant human P2X7 responses

P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1β (IL-1β) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC₅₀ of 24 μM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC₅₀ of 6.4 μM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1β secretion from lipopolysaccharide (LPS)-primed human CD14⁺ monocytes was suppressed with trifluoperazine and paroxetine.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

α-Factor inhibition of the rate of cell passage through the “start” step of cell division in Saccharomyces cerevisiae yeast: Estimation of the division delay per α-factor·receptor complex

A highly sensitive, kinetically unambiguous assay for α-factor-induced delay of cell passage through the “start” step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the α-factor execution point of start. The t_1/2 for cell passage through start by this population of cells is 31 min in the absence of α-factor. The inhibition constant, K_I, represents the α-factor concentration which produces a 50% inhibition of this rate and is equal to 2×10⁻¹⁰M. A second assay for maximal cell division arrest by α-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125±5 h at saturating α-factor, and a K₅₀ (that is, an α-factor concentration which produces a half-maximal response) of 2.5×10⁻⁸M. Both assays were performed in the effective absence of α-factor inactivation. Values of the dissociation constant K_D and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5×10⁻⁸M and 10⁴, respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor·α-factor complex which is present on the cell at equilibrium producing a 43±10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to α-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM α-factor to a 125±5 h maximum arrest at saturating α-factor concentrations of >170 nM. The possible significance of this observation toward the mechanism of α-factor-induced cell division arrest is discussed.     

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol have been determined by single-crystal X-ray diffraction studies. The space group of the α-cyclodextrin–p-chlorophenol complex is P2₁2₁2₁ with unit cell dimensions of a=15.299(3), b=24.795(5), c=13.447(5) Å, and that of the α-cyclodextrin–p-cresol complex is P2₁ with unit cell dimensions of a=7.927(7), b=13.568(7), c=24.54(1) Å, β=90.41(8)°. In spite of the similar structures of guest molecules, both complexes have different inclusion modes and packing structures.     

Expression of the Mucosal Homing Receptor α4β7 Correlates with the Ability of CD8+ Memory T Cells To Clear Rotavirus Infection

The integrin α₄β₇ plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with α₄β₇ expression. α₄β₇^hi memory phenotype (CD44^hi), α₄β₇⁻ memory phenotype, and presumptively naive (CD44^lo) CD8⁺ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. α₄β₇^hi memory phenotype CD8⁺ cells were highly efficient at clearing rotavirus infection, α₄β₇⁻ memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44^lo cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor α₄β₇.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.     

A novel, convenient assay of lanosterol 14α-demethylase

A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed. Three substrates, [32-³H]-24,25-dihydrolanosterol, [32-³H]lanost-8-en-3β,32-diol, and [32-³H]lanost-7-en-3β-32-diol, were studied. In all cases, the rate of tritium released into aqueous solution provided a simple and direct assay of 14α-demethylase activity. The kinetic parameters of K_m and V_max for each substrate have been determined in a reconstituted system from rat liver. The percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods. Separation of unreacted sterol from tritiated formate and water via reverse-phase chromatography permitted several samples to be analyzed at once.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Anion inhibition studies of the Zn(II)-bound ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO₂ to bicarbonate and protons, with a k_cat value of 3.0 × 10⁵ s⁻¹ and k_cat/K_M value of 3.9 × 10⁷ M⁻¹ s⁻¹. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (K_I values of 6.2–94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (K_I values of 0.71–0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed K_I values in the range of 3.1–9.3 mM.     

Direct and Regulated Interaction of Integrin αEβ7 with E-Cadherin

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, α_Eβ₇, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant α_Eβ₇, and to α_Eβ₇ solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY′ cells expressing the α_Eβ₇ integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to α_Eβ₇ or E-cadherin.

The binding of α_Eβ₇ integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through α_Eβ₇ requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the α_E-chain is unique among integrins, the avidity of α_Eβ₇ for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of α_Eβ₇ for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the α_Eβ₇ integrin.

     

Structures, dynamic behaviour in solution and cross-coupling reactions of the α-palladiated (η-alkylarene)tricarbonylchromium complexes containing a palladium-chromium bond

Structures of the complexes (η³-C₃H₅)Pd(μ-η^6:1-CH₂PhCr(CO)₃ and (η³-C₃H₅)Pd[μ-η^6:1-CH(Ph)Ph]Cr(CO)₃ in solution were evaluated by NMR (¹H and ¹³C) and IR spectroscopy. The dynamic behaviour of the complexes was investigated. Quick rotation (on the NMR time scale) of the tricarbonylchromium groups around the axis passing through the centre of the η⁶-coordinated phenyl ring and the chromium atom takes place at room temperature and becomes slow on cooling. The η³-allylic ligand was proved to undergo no dynamic changes in solution. Unlike the solid state, the semi-bridging carbonyl groups between chromium and palladium atoms are absent or very weak in solution. Cross-coupling reactions of the complexes with organohalides are described.