Alkylated poly(amino acids). I. Conformational properties of poly(Nε-trimethyl-L-lysine) and poly(Nδ-trimethyl-L-ornithine)

Poly(N^ε-trimethyl-L -lysine), [Lys(Me₃)]_n, and poly(N^δ-trimethyl-L -ornithine), [Orn(Me₃)]_n, in sodium dodecylsulfate do not assume the β-structure or α-helix, respectively, of their parent polymers. In 0.5M Ca(ClO₄)₂ both [Lys(Me₃)]_n and [Orn(Me₃)]_n are aggregated and display CD spectra indicative of a regular, perhaps helical, structure. For [Lys]_n and [Lys(Me₃)]_n, the T₁ of the α-hydrogens are 0.379 and 0.230 sec, respectively, indicating greater rigidity for [Lys(Me₃)]_n. The CD spectrum of [Lys(Me₃)]_n at pH 8 is more heat resistant than that of [Lys]_n. It is suggested that apolar interactions are more important in the methylated polymers than in the parent polymers.     

Helix promotion in polypeptides by polyols

The helix content of [L -Lys(Me₃)]_n · ClO₄, and [L -Lys(Me₃)⁵⁰, L -Ala⁵⁰]_n · ClO₄ in water is markedly increased by the presence of sucrose and glycerol. For [L -Lys(Me₃)]_n · ClO₄ the ellipticity at 222 nm changes from +2 × 10³ deg cm² dmole^?1 in water to ?44 × 10³ in 50% glycerol. Sucrose does not promote helix formation in melittin at pH 7.2, but glycerol does. At pH 5.5 sucrose and, more so, glycerol, induce helix in melittin. Glycerol induces some helix in methylated melittin, but less than in melittin. The results are discussed in relation to excluded volume effects, ΔG of transfer of peptide and hydrophobic groups from water to mixed solvents, electrostatic effects, and preferential hydration. © 1993 John Wiley & Sons, Inc.     

Titanium, zirconium and hafnium halide complexes of trithioether and triselenoether ligands

The reaction of TiX₄(X=Cl or Br) with the tripodal ligands MeC(CH₂SMe)₃ or MeC(CH₂SeMe)₃, (L³) in anhydrous n-hexane or CH₂Cl₂ produced the extremely moisture sensitive complexes [TiX₄(L³)]. These were characterised by microanalysis, IR, UV-Vis and variable temperature ¹H,¹³C{¹H} and ⁷⁷Se NMR spectroscopy. The NMR studies showed that in solution in CH₂Cl₂ the complexes contain L³ bound as bidentates, and that pyramidal inversion and exchange between the free and coordinated chalcogen donors is rapid at room temperature. Ligand dissociation/exchange increases TiCl₄<TiBr₄ and MeC(CH₂SMe)₃<MeC(CH₂SeMe)₃ and attempts to isolate TiI₄ analogues were unsuccessful. The reactions of [MCl₄(Me₂S)₂] (M=Zr or Hf) with (L³) in anhydrous CH₂Cl₂ produces white or cream 7-coordinate [MCl₄(L³)], which are insoluble in chlorocarbon solvents. The reactions of TiX₄ (X=Cl, Br or I) with the trithia-macrocycles [9]aneS₃ and [10]aneS₃ produced [TiX₃([n]aneS₃)]X, whilst reaction of TiCl_4, SbCl₅ and [9]aneS₃ in anhydrous CH₂Cl₂ gave [TiCl₃([9]aneS₃)]SbCl₆. Spectroscopic studies suggest these macrocyclic compounds contain 6-coordinate cations, [TiX₃([n]aneS₃)]⁺ (n=9 or 10) but with Zr and Hf the complexes [MCl₄([n]aneS₃)] are 7-coordinate and neutral.     

Reaction of platinum(II) diamine and triamine complexes with selenomethionine

We have reacted [Pt(dien)Cl]Cl, [Pt(en)(D₂O)₂]²⁺, and [Pt(Me₄en)(D₂O)₂]²⁺ [Me₄en = N,N,N′,N′-tetramethylethylenediamine] with selenomethionine (SeMet). When [Pt(dien)Cl]Cl is reacted with SeMet, [Pt(dien)(SeMet-Se)]²⁺ is formed; two Se-CH₃ resonances are observed due to the different chiralities at the Se atom upon platination. In a reaction of [Pt(dien)Cl]Cl with an equimolar mixture of SeMet and Met, the SeMet product forms more quickly though a slow equilibrium with approximately equal amounts of both products is reached. [Pt(Me₄en)(D₂O)₂]²⁺ reacts with SeMet to form [Pt(Me₄en)(SeMet-Se)(D₂O)]²⁺ initially but forms [Pt(Me₄en)(SeMet-Se,N)]⁺ ultimately. One stereoisomer of the chelate, assigned to the R chirality at the Se atom, dominates within the first few minutes of reaction. [Pt(en)(D₂O)₂]²⁺ forms a variety of products depending on reaction stoichiometry; when one equivalent or less of SeMet is added, the dominant product is [Pt(en)(SeMet-Se,N)]⁺. In the presence of excess SeMet, [Pt(en)(SeMet-Se)₂]²⁺ is the dominant initially, but displacement of the en ligand occurs leading to [Pt(SeMet-Se,N)₂] as the eventual product. Displacement of the en ligand from [Pt(en)(SeMet-Se,N)]⁺ does not occur. In reactions of K₂PtCl₄ with two equivalents of SeMet, [Pt(SeMet-Se,N)₂] is formed, and three sets of resonances are observed due to different chiralities at the Se atoms. Only the cis geometric isomers are observed by ¹H and ¹⁹⁵Pt NMR spectroscopy.     

Antimicrobial,cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH₂), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp⁴→Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 μM) compared with the naturally occurring peptide. The substitution Ala¹⁸→Lys and the double substitution Asp⁴→Lys and Ala¹⁸→Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC₅₀ values in the range of 12–20 μM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC₅₀ = 20 μM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca²⁺] in BRIN-BD11 cells when incubated at a concentration of 1 μM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.     

Synthesis and biological evaluation of tricarbonyl Re(I) and Tc(I) complexes anchored by poly(azolyl)borates: application on the design of radiopharmaceuticals for the targeting of 5-HT<Subscript>1A</Subscript> receptors

The building blocks fac-[^99mTc{κ³-HB(tim^Me)₃}(CO)₃] and fac-[^99mTc{κ³-R(μ-H)B(tim^Me)₂}(CO)₃] [R is H (4a), Ph (5a); tim^Me is 2-mercapto-1-methylimidazolyl] were obtained almost quantitatively by reacting fac-[^99mTc(CO)₃(H₂O)₃]⁺ with the corresponding scorpionate. These compounds cross the intact blood–brain barrier in mice, with significant retention in the case of 4a and 5a. Using 4a as the lead structure, we have synthesized the functionalized complexes fac-[M{κ³-H(μ-H)B(tim^Bu-pip)₂}(CO)₃] [M is Re (8), ^99mTc (8a); tim^Bu-pip is methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl](2-mercapto-1-methylimidazol-5-yl)methanamide] and fac-[M{κ ³-H(μ-H)B(tim^Me)(tim^Bu-pip)}(CO)₃] [M is Re (9), ^99mTc (9a)] and evaluated their potential as radioactive probes for the targeting of brain 5-HT_1A serotonergic receptors. The Re complexes exhibit excellent affinity [IC₅₀=0.172 ± 0.003 nM (8); IC₅₀=0.65 ± 0.01 nM (9)] for the 5-HT_1A receptor. The radioactive congeners (^99mTc) have shown an initial brain uptake of 1.38 ± 0.46%ID g⁻¹ (8a) and 0.43 ± 0.12%ID g⁻¹ (9a), but suffer from a relatively fast washout.     

Oxidation of [Ir(η-C5Me5)(C3S5)] [C3S5 = 4,5-disulfanyl-1,3-dithiole-2-thionate(2−)] and X-ray crystal structure of [IrBr(η-C5Me5)(μ-C2S4)IrBr(η-C5Me5)]

[Ir(η⁵-C₅Me₅)(C₃S₅)] [C₃S₅²⁻ = 4,5-disulfanyl-1,3-dithiole-2-thionate(2−)] was prepared by a reaction of [NMe₄]₂[C₃S₅] with [Ir(η⁵- C₅Me₅)Cl₂]₂ in ethanol. It was reacted with bromine to afford a paramagnetic species [IrBr(η⁵-C₅Me₅)(C₃S₅)] with the Ir-Br bond and in the one-electron-oxidized state, and a diamagnetic dinuclear species [IrBr(η⁵-C₅Me₅)(μ-C₂S₄)IrBr(η⁵-C₅Me₅)]. ESR spectra for the one-electron-oxidized species in solution are discussed. The X-ray crystal structural analysis for the latter complex revealed the geometry consisting of dinuclear IrBr(η⁵-C₅Me₅) moieties bridged by the C₂S₄²⁻ ligand.     

13C Nuclear magnetic resonance study of salt induced conformational change of basic polypeptides: Poly (L-lysine), poly (L-arginine), and poly (L-ornithine)

A ¹³C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)_n], poly(L -arginine) [(Arg)_n], and poly (L -ornithine) [(Orn)_n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the C^α and carbonyl carbon signals are observed in the helical state. The upfield shift of the C^β signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)_n and (Arg)_n. However, the displacement of the C^α, C^β, and carbonyl carbons of (Orn)n in the presence of 4M NaClO₄ is found to be almost 50% of that of (Lys)_n and (Arg)_n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T₁), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)_n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)_n, however, the precipitate of the helical polymers is mainly due to aggregation.     

Study of salt effects on adenosine–polyuridylic acid interaction

Complex formation between poly(U) and adenosine in solutions of salts that stabilize (Na₂SO₄), destabilize (NaClO₄), or have little effect on the water structure (NaCl), as well as the poly(U)·poly(A) interaction in NaClO₄, was studied by equilibrium dialysis and uv spectroscopy. At 3°C and neutral pH, Ado·2 poly(U) is formed in 1M NaCl and 0.33M Na₂SO₄. In NaClO₄ solutions under the same conditions, an Ado·poly(U) was found over the whole range of salt concentration investigated (10 mM?1M), which has not been previously observed under any conditions. The Ado-poly(U) was also found in a NaCl/NaClO₄ mixture, the transition from the triple- to the double-helical complex occurring within a narrow range of concentration of added NaClO₄. In the presence of 1M NaCl this transition is observed on adding as little as 10 mM NaClO₄, i.e., at a [ClO]/[Cl^?] ratio of about 1:100. However, when NaClO₄ is added to a 1M solution of the stabilizing salt Na₂SO₄, no transition occurs even at a [ClO]/[SO] ratio of 1:4. Investigation of melting curves and uv spectra has shown that in an equimolar mixture of the polynucleotides, only a double-helical poly(U)·poly(A) exists in 1M NaClO₄ at low temperatures; this also holds for 1M NaCl. This changes to a triple-helical 2 poly(U)·poly(A) and then dissociates as the temperature increases. At low temperatures and the poly(U)/poly(A) concentration ratio of 2:1, a mixture of 2 poly(U)·poly(A) and poly(U)·poly(A) was observed in 1M NaClO₄, in contrast to the case of 1M NaCl. Thus, sodium perchlorate, a strong destabilizer of water structure, promotes formation of double-helical complexes both in the polynucleotide–monomer and the polynucleotide–polynucleotide systems. Beginning with a sufficiently high ionic strength (μ ? 0.9), a further increase in the salt molarity results in an increase of the poly(U)·adenosine melting temperature in both stabilizing and neutral salts and a decrease in the destabilizing salt. In Na₂SO₄ concentrations higher than 1.2M Ado·2 poly(U) precipitates at room temperature. Analysis of the binding isotherms and melting profiles of the complexes between poly(U) and adenosine according to Hill's model shows that the cooperativity of binding, due to adenosine stacking on poly(U), increases in the order NaClO₄ < NaCl < Na₂SO₄. The free energy of adenosine stacking on the template is similar to that of hydrogen bonding between adenosine and poly(U) and ranges from ?1 to ?2 kcal/mol. The values of ΔH_t [the effective enthalpy of adenosine binding to poly(U) next to an occupied site, obtained from the relationship between complex melting temperature and free monomer concentration at the midpoint of the transition] are ?14.2, ?18.3, and ?16.8 kcal/mol for 1M solutions of NaClO₄, NaCl, and Na₂SO₄, respectively. The results indicate that the effects of anions of the salts studied are related to water structure alterations rather than to their direct interaction with the complexes between poly(U) and adenosine.     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Phosphorus-carbon bond formation via reactions of triphenylphosphine with acetylene and pentamethylcyclopentadienyl coordinated to iridium(III)

Phosphorus-carbon bond is formed via: (i) the apparent HCCH insertion into Ir-P bond to produce Ir-CHCH-PPh₃ group and (ii) the activation of the ring-methyl group of the coordinated Cp^* (C₅Me₅ ⁻) to produce Ir(η⁵-C₅Me₄CH₂-PPh₃) group from reactions of iridium(III)-Cp^* complexes, [Cp^*IrL₃]ⁿ⁺ (n=1, 2); Cp^*=C₅Me₅ ⁻; L₃=Cl(PPh₃)₂ (3), (CH₃CN)₃ (5). The following new P-C bond containing iridium(III) complexes have been prepared: [Cp^*Ir(-CHCH-PPh₃)Cl(PPh₃)]⁺ (4) from 3 with HCCH; [Ir(η⁵-C₅Me₄CH₂-PPh₃)(H)(PPh₃)₂]²⁺ (6) from 5 with PPh₃; [Cp^*Ir(-CHCH-PPh₃)₂(PPh₃)]²⁺ (7) from 5 with HCCH and PPh₃; [Ir(η⁵-C₅Me₄CH₂-PPh₃)(-CHCH-PPh₃)Cl(PPh₃)]²⁺ (8) from [Ir(η⁵-C₅Me₄CH₂-PPh₃)(Cl)(PPh₃)₂]²⁺ (6-Cl) with HCCH; [Ir(η⁵-C₅Me₃(1,3-CH₂-PPh₃)₂(H)(PPh₃)₂)]³⁺ (10) from [Ir(η⁵-C₅Me₄CH₂-PPh₃)(NCCH₃)₂(PPh₃)]³⁺ (9) with PPh₃; [Ir(η⁵-C₅Me₄CH₂-PPh₃)(-CHCH-PPh₃)₂(PPh₃)]³⁺ (11) from 9 with HCCH and PPh₃.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Predicting responses of photosynthesis and root fraction to elevated [CO2]a: interactions among carbon,nitrogen, and growth*

At elevated atmospheric CO₂ concentrations ([CO₂]_a), photosynthetic capacity (A_max) and root fraction (η_R, the ratio of root to plant dry mass) increased in some studies and decreased in others. Here, we have explored possible causes of this, focusing on the relative magnitudes of the effects of elevated [CO₂]_a on specific leaf (n_m) and plant (n_p) nitrogen concentrations, leaf mass per unit area (h), and plant nitrogen productivity (α). In our survey of 39 studies with 35 species, we found that elevated [CO₂]_a led to decreased n_m and n_p in all the studies and to increased h and α in most of the studies. The magnitudes of these changes varied with species and with experimental conditions. Based on a model that integrated [CO₂]_a-induced changes in leaf nitrogen into a biochemically based model of leaf photosynthesis, we predicted that, to a first approximation, photosynthesis will be upregulated (A_max will increase) when growth at increased [CO₂]_a leads to increases in h that are larger than decreases in n_m. Photosynthesis will be downregulated (A_max will decrease) when increases in h are smaller than decreases in n_m. The model suggests that photosynthetic capacity increases at elevated [CO₂]_a only when additional leaf mesophyll more than compensates the effects of nitrogen dilution. We considered two kinds of regulatory paradigms that could lead to varying responses of η_R to elevated [CO₂]_a, and compared the predictions of each with the data. A simple static model based on the functional balance concept predicts that η_R should increase when neither n_p nor h is very responsive to elevated [CO₂]_a. The quantitative and qualitative agreement of the predictions with data from the literature, however, is poor. A model that predicts η_R from the relative sensitivities of photosynthesis and relative growth rate to elevated [CO₂]_a corresponds much more closely to the observations. In general, root fraction increases if the response of photosynthesis to [CO₂]_a is greater than that of relative growth rate.     

Syntheses, structures and spectroscopy of new heteroselenometalates

[PPh₄]₂[MoSe₄] and [PPh₄]₂[WSe₄] react with two equivalents of AuCN in CH₃CN to afford [PPh₄]₂[(NC)Au(μ-Se)₂-Mo(μ-Se)₂Au(CN)] · CH₃CN (bd1) and [PPh₄]₂[(NC)Au(μ-Se)₂Au(CN)] · CH₃CN (bd2), respectively. Compounds 1 and 2 are isostructural. Compound 1 crystallizes in the triclinic space group with two formula units in a cell dimensions a=13.181(4), b=14.239(4), C=14.684(4) Å, α=73.00(3), β=73.66(2), γ=79.06(2)° at 113 K. Full anisotropic refinement of the structure of 1 on F² led to a value of R₁=0.0562 for those 9075 data having F_o² . 2σ(F_o²). The [(NC)Au(μ-Se)₂Mo(μ-Se)₂Au(CN)]²⁻ anion of 1 comprises two AuCN fragments ligating the opposite edges of a tetrahedral [MoSe₄]²⁻ moiety. Compounds 1 and 2 exhibit an IR band at 2250 cm⁻¹ that may be assigned to a CN stretching mode of the CH₃CH solvate; such a solvate molecule was found in the crystal structure of 1. The ⁷⁷Se NMR spectra show a resonance at 1104 ppm for 1 and 832 ppm for 2. Addition of excess PME₂Ph to the same solutions that produce 1 and 2 results in the formation of [PPh₄][(Me₂PhP)Au(μ-Se)₂MoSe₂] (3) and [PPh₄][(Me₂PhP)Au(μ-Se)₂WSe₂] (4), respectively. Compounds 3 and 4 are not isostructural. Compound 3 crystallizes in the monoclinic space gorup Cc with four formula units in a cell of dimensions a=21.912(4), B=9.809(2), C=15.959(3) Å, β=100.79(3) at 113 K. Full anisotropic refinement of the structure of 1 on F² led to a value of R₁=0.0481 for those 6851 data having F_o² > 2σ(F_o²). Compound 4 crystallizes in the triclinic space group with two formula units in a cell of dimensions a=11.315(2), B=13.053(3), C=14.173(3) Å, α=103.59(3), β=103.55(3), γ=114.75(3)δ at 113 K. Full anisotropic refinement of the structure of 4 on F² led to a value of R₁=0.0414 for those 7825 data having F_o² > 2σ(F_o²). The [(Me₂]PhP)Au(μ-Se)₂MoSe₂]⁻ anion of 3 and the [(Me₂PhP)Au(μ-Se)₂WSe₂]⁻ anion of 4 comprise an [(Me₂PhP)Au]⁺ fragment ligated across an edge of a tetrahedral (MSe₄]²⁻ moiety. [PPh₄]₂[MoSe₄] and [PPh₄]₂[WSe₄] react with one equivalent of AuCN in CH₃CH to afford [PPh₄]₂[(NC)Au(μ-Se)₂MoSe₂] (5) and [PPh₄]₂[(NC)Au(μ-Se)₂WSe₂] (6), respectively. Compounds 5 and 6 are isostructural. Compounds 5 crystallizes in the monoclinic space group C2/c with four formula units in a cell of dimensions a=11.234(15), B=20.329(28), C=20.046(28) Å, β=91.81(5)° at 113 K. Full anisotropic refinement of the structure of 5 on F² led to a value of R₁=0.0457 for those 4003 data having F_o² > 2σ(F_o²). The [(NC)Au(μ-Se)₂MoSe₂]²⁻ anion of 5, which has a crystallographically imposed twofold axis, comprises an AuCN fragment ligated across an edge of a tetrahedral [MoSe₄]²⁻ moiety. The reaction of [PPh₄]₂(NC)Cu(μ-Se)₂MoSe₂] with one equivalent of AuCN in CH₃CN produces a precipitate that is then redissolved through reaction with an excess of PMe₂Ph to afford [PPh₄][(Me₂PhP)₂Cu(μ-Se)₂MoSe₂] (7). Compound 7 crystallizes in the monoclonic space group P1₁/c with four formula units in a cell of dimensions a=9.975(1), B=30.391(7), C=14.541(6) Å, β=109.66(3) at 113 K. Full anisotropic refinement of the structure of 7 on F² led to a value of R₁=0.0305 for those 5205 data having F_o² > 2σ(F_o²). The [(Me₂PhP)₂Cu(μ-Se)₂MoSe₂]⁻ anion of 7 comprises an [(Me₂PhP)₂Cu]⁺ fragment ligated across an edge of the [MoSe₄]²⁻ moiety to provide a tetrahedral geometry about the Cu atom. The NMR, IR and UV-Vis spectroscopic data for these compounds are consistent with their solid-state structures.     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

UV-Vis and fluorimetric Al, Zn, Cd and Pb complexation studies of two 3-hydroxyflavones and a 3-hydroxythioflavone

The complexation of Al³⁺, Zn²⁺, Cd²⁺ and Pb²⁺ by the 3-hydroxyflavones: 3-hydroxy-2-(2-methoxyphenyl)-4H-1-benzopyran-4-one (H1) and 3-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one (H2), and by the 3-methoxythioflavone: 3-hydroxy-2-(2-methoxyphenyl)-4H-1-benzopyran-4-thione (H3) have been studied spectrophotometrically and fluorimetrically to determine the corresponding complexation constants, K_sp and K_fl, in 5:95 water:ethanol (v/v) solution for which [HClO₄] was either 10⁻² or 10⁻⁵ mol dm⁻³ and I = 0.10 mol dm⁻³ (NaClO₄) at 298.2 K. Complexation occurs dominantly through the deprotonated ligand for [Al(1)]²⁺ and [Al(2)]²⁺ for which log K_sp = 4.51 and 4.73, respectively, in 10⁻² mol dm⁻³ HClO₄ and 4.21 and 4.61 in 10⁻⁵ mol dm⁻³ HClO₄. For Pb²⁺ complexation by H1, H2 and H3 is characterized by log K_sp = 2.20, 2.57 and 3.22, respectively, in 10⁻² mol dm⁻³ HClO₄ and 4.70, 5.38 and 5.74 in 10⁻⁵ mol dm⁻³ HClO₄. Equilibrium mixtures of [Pb(H1)]²⁺ and [Pb1]⁺, [Pb(H2)]²⁺ and [Pb2]⁺, and [Pb(H3)]²⁺ and [Pb3]⁺ appear to be formed. Complexation of Zn²⁺ and Cd²⁺ by all three ligands was only detected in 10⁻⁵ mol dm⁻³ HClO₄. For Zn²⁺ complexation by H1, H2 and H3 log K_sp = 3.22, 3.74 and 4.46 and for Cd²⁺ the corresponding values are 2.39, 2.40 and 3.72 for Cd²⁺. Only [Al1]²⁺ and [Al2]²⁺ show significant fluorescence and are characterized by log K_fl = 6.30 and 7.49 in 10⁻² mol dm⁻³ HClO₄.     

A fluorescence study of differently substituted 3‐styrylindoles and their interaction with bovine serum albumin

Interaction of 3‐styrylindoles 1–8 viz. 3‐(2‐phenylethenyl‐E)‐NH‐indole (1), 3‐[2‐(4‐nitrophenyl)ethenyl‐E]‐NH‐indole (2), 5‐bromo‐3‐[2‐(4‐nitrophenyl)ethenyl‐E]‐NH‐indole (3), 5‐methoxy‐3‐[2‐(4‐nitrophenyl)ethenyl‐E]‐NH‐indole (4), 3‐[2‐(4‐cyanophenyl)ethenyl‐E]‐NH‐indole (5), 3‐[2‐(4‐cyanophenyl)ethenyl‐E]‐N‐ethylindole (6), 5‐bromo‐3‐[2‐(4‐chlorophenyl)ethenyl‐E]‐NH‐indole (7) and 5‐methoxy‐3‐[2‐(4‐chlorophenyl)ethenyl‐E]‐NH‐indole (8) with bovine serum albumin (BSA) was examined by UV–vis and steady‐state fluorescence spectroscopy. The fluorescence intensity of 1–8 increases with the increasing BSA concentration. Upon binding with BSA, while 1 and 5–8 show a blue shift in their λ_{f max}, 2–4 do not exhibit such behavior. Compounds 1–8 also quench the 345 nm fluorescence of BSA in phosphate buffer (λ_ex, 280 nm). These compounds intercalate in the hydrophobic regions of BSA, as evidenced by the determination of BSA binding site micropolarity using compounds 2–8. As evidenced by the estimation of energy transfer efficiency and distance between the donor (BSA‐Trp‐212) and the acceptor (3‐styrylindoles), the halo‐substituted compounds 3 and 7 interact with BSA more effectively than the other 3‐strylindoles. These compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments in proteins, polymers, micelles, etc. Copyright © 2008 John Wiley & Sons, Ltd.     

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca

We have studied the modulation by intracellular Ca²⁺of the epithelial Ca²⁺channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca²⁺measurements:1. Currents through ECaC were dramatically inhibited if Ca²⁺was the charge carrier. This inhibition was dependent on the extracellular Ca²⁺concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca²]_einduced in non-Ca²] buffered HEK 293 cells at −80 m V an increase in intracellular Ca²⁺([Ca²]_i) with a maximum rate of rise of 241 ±15nM/s (n= 18 cells) and a peak value of 891 ± 106 nM. The peak of the concomitant current with a density of 12.3 ± 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca²⁺transient, as expected if the Ca²⁺transient is due to influx of Ca²⁺. Consequently, no Ca²⁺] signal was observed in cells transfected with the Ca²⁺impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca²⁺]_iby dialyzing the cell with pipette solutions containing various Ca²⁺] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na⁺or Ca²⁺] ions. Half maximal inhibition of Ca²⁺currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na⁺currents in the absence of Ca²⁺] and Mg²⁺were inhibited with an IC₅₀of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca²⁺and Na⁺, which are mainly carried by Ca²⁺, are inhibited by [Ca²]_iwith an IC₅₀of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca²⁺impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca²]_i(IC₅₀= 123 nM, n between 7 and 18).4. The sensitivity of ECaC currents in inside-out patches for [Ca²]_iwas slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na⁺was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca²]_e(n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca²⁺(whole-cell configuration) or removal of Ca²⁺from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 ± 34 s (n= 15) in whole-cell mode and after 135 ± 23 s (n= 17) in inside-out patches.6. We conclude that influx of Ca²⁺through ECaC and [Ca²]_iinduce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca²⁺in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca²⁺from an internal Ca²⁺binding site at ECaC.     

Measurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method

A method for the measurement of the cytosolic Na⁺ concentration in intact synaptosomes is described. This method makes use of a pH sensitive dye (BCECF) that can be loaded into the cytosol and a relatively specific ionophore (monensin) that can exchange Na⁺ for H⁺ across the synaptosomal membrane. By setting conditions such that there is no electrochemical potential difference for H⁺ across the membrane (no membrane potential and pH_i = pH_o), addition of ionophore would induce a H⁺ flux only if there is a concentration difference for Na⁺. Thus, when there is no fluorescence change (no cytosolic pH change) extracellular [Na⁺] equals intrasynaptosomal [Na⁺. The intrasynaptosomal [Na⁺] concentration was determined to be 7 ± 3 mM (n = 5; mean ± S.E.). The results obtained with this fluorescence method are compared with estimates obtained by atomic absorption spectrometry. Limitations and applications of the method are discussed.     

Inorganic-carbon uptake by the marine diatom Phaeodactylum tricornutum

Air-grown cells of the marine diatom Phaeodactylum tricornutum showed only 10% of the carbonic-anhydrase activity of air-grown Chlamydomonas reinhardtii. Measurement of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in Phaeodactylum. Photosynthetic oxygen evolution at constant inorganic-carbon concentration but varying pH showed that exogenous CO₂ was poorly utilized by the cells. Sodium ions increased the affinity of Phaeodactylum for HCO ₃ ^- and even at high HCO ₃ ^- concentrations sodium ions enhanced HCO ₃ ^- utilization. The internal inorganic-carbon pool (HCO ₃ ^- +CO₂] was measured using a silicone-oil-layer centrifugal filtering technique. The internal [HCO ₃ ^- +CO₂] concentration never exceeded 15% of the external [HCO ₃ ^- +CO₂] concentration even at the lowest external concentrations tested. It is concluded that an internal accumulation of inorganic carbon relative to the external medium does not occur in P. tricornutum.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid