microM Range Research Articles

Gold(III) complexes as potential antitumor agents: solution chemistry and cytotoxic properties of some selected gold(III) compounds.

Gold(III) complexes generally exhibit interesting cytotoxic and antitumor properties, but until now, their development has been heavily hampered by their poor stability under physiological conditions. To enhance the stability of the gold(III) center, we prepared a number of gold(III) complexes with multidentate ligands - namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2), and [Au(phen)Cl(2)]Cl - and analyzed their behavior in solution. The solution properties of these complexes were monitored by visible absorption spectroscopy, mass spectrometry, and chloride-selective potentiometric measurements; the electrochemical properties were also studied by cyclic voltammetry and coulometry. Since all the investigated compounds exhibited sufficient stability under physiological conditions, their cytotoxic properties were tested in vitro, via the sulforhodamine B assay, on the representative human ovarian tumor cell line A2780, either sensitive or resistant to cisplatin. In most cases the investigated compounds showed relevant cell-killing properties with IC(50) values falling in the 0.2-10 microM range; noticeably most investigated gold(III) complexes were able to overcome, to a large extent, resistance to cisplatin when tested on the corresponding cisplatin-resistant cell line. The cytotoxic properties of the free ligands were also determined under the same solution conditions. Ethylenediamine, diethylenetriamine, and cyclam were virtually nontoxic (IC(50) values > 100 microM) so that the relevant cytotoxic effects observed for [Au(en)(2)]Cl(3) and [Au(dien)Cl]Cl(2) could be quite unambiguously ascribed to the presence of the gold(III) center. In contrast the phenanthroline and terpyridine ligands turned out to be even more cytotoxic than the corresponding gold(III) complexes rendering the interpretation of the cytotoxicity profiles of the latter complexes less straightforward. The implications of the present findings for the development of novel gold(III) complexes as possible cytotoxic and antitumor drugs are discussed.

3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth.

Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.

Detection of short-lived electrogenerated species by Raman microspectrometry

Raman microprobe spectrometry has been applied to the characterization of unstable species generated electrochemically at a microelectrode (radius in the 10 microm range). The ability of the spectroelectrochemical method to detect short-lived intermediates is directly related to its capability to probe small volumes. Raman microprobe spectrometry is appropriate for electrochemical applications because it allows the analysis of approximately 1 microm3 of solution. In spectroelectrochemical experiments, such a volume corresponds to a reaction layer of 1 microm thickness. Potentially, this technique can allow the observation of species with lifetimes of the order of 1 ms. To enhance the capabilities of this spectroscopic technique, we utilized it in combination with steady-state voltammetry at a microelectrode, to increase the concentration of unstable intermediates near the electrode surface. To determine the detection limit of this combined technique, we varied the base concentration as a means for varying the lifetime the radical cation electrogenerated from 9,10-dichloroanthracene. Well-resolved resonance Raman spectra were obtained for this radical cation when the lifetime was > or = 0.1 ms. This short time resolution achieved with micro-Raman spectroelectrochemistry makes this technique a powerful tool for the characterization of short-lived intermediates that are generated electrochemically in solution.

Antibacterial and Antifungal Activities of Vasostatin-1, the N-terminal Fragment of Chromogranin A

Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 microM range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized. In addition, from the comparison with the activity displayed by related peptides (human recombinant and rat synthetic fragments), we could determine that antibacterial and antifungal activities have different structural requirements. To assess for such activities in vivo, CGA and CGA-derived fragments were identified in secretory material released from human polymorphonuclear neutrophils upon stimulation. Vasostatin-1, which is stored in a large variety of cells (endocrine, neuroendocrine, and neurons) and which is liberated from stimulated chromaffin and immune cells upon stress, may represent a new component active in innate immunity.

Tumor apoptosis induced by epoxide-containing piperazines, a new class of anti-cancer agents

The overall purpose of this study was to determine the potential efficacy of epoxide-containing piperazines as a new class of anti-cancer agents. Two representative compounds, specifically NCO-700, a 4-trimethoxyphenyl-substituted epoxide-piperazine, and TOP-008, a 4-phenylpropenyl-substituted epoxide-piperazine were tested in cytotoxic assays with human breast and prostate cancer cell lines. A second objective was to determine if these two compounds had anti-cancer activity in vivo when tested against xenograft tumors in nude mice or human tumors grown under the kidney capsule in mice. A final objective of this study was to establish if NCO-700 and TOP-008 achieved cancer cell killing through an apoptotic mechanism. The anti-proliferative activity of NCO-700 and TOP-008 were tested in a 7 day cell-survival assay utilizing a number of well characterized breast (HS-578T, T47D, MCF-7) and prostate (DU-145, PC-3, LNCaP) cancer cell lines. In vivo studies with the two compounds were performed, in nude mice bearing DU-145 xenograft tumors, and in normal mice in which DU-145 prostate cancer cells and HS-578T breast cancer cells were grown as solid tumors in the subrenal capsules of the animals. Apoptotic cell death of cancer cells was determined by a number of established techniques that detect apoptosis, including the confocal laser microscopy of treated cells and mitochondrial leakage assays utilizing the cationic dye, JC-1. Finally, the activation of the caspase cascade, enzymes that carry out apoptosis in mammalian cells, was examined in treated cells by immunoblot assays. NCO-700 and TOP-008 displayed cytotoxicity to HS-578T human breast cancer cells, with ED(50) values in the 3-6 microM range. Cytotoxicity to androgen receptor-negative human prostate cancer cells (PC-3 and DU-145 cells) occurred with ED(50) values in the 5-20 microM range. Cytotoxicity to hormone receptor-positive breast and prostate cancer cell lines occurred at 10 to 20-fold higher concentrations of the two compounds. When human prostate (DU-145) or breast cancer (HS-578T) cells were grown as solid tumors in the subrenal capsules of mice, significant anti-tumor activity of NCO-700 was observed at 20 mg/kg and 50 mg/kg body weight respectively, for prostate and breast tumors. In nude mice bearing DU-145 prostate tumor xenografts, 50 mg/kg doses of the two compounds either stopped (TOP-008) tumor growth or slowed (NCO-700) growth. The mechanism of cytotoxicity was shown to be through apoptosis, (a) by confocal microscopy studies revealing nuclear fragmentation, (b) by mitochondrial studies revealing disruption of the mitochondrial membrane and release of the cationic dye, JC-1, into the cytoplasm and (c) by protein immunoblot assays indicating that over a 6 h period, TOP-008 induced a significant accumulation of the pro-apoptotic protein, bak, in the mitochondrial fraction of HS-578T human breast cancer cells, accompanied by activation, at 2.5 h, of caspase-3. These studies indicated that the epoxide-containing piperazines, as exemplified by NCO-700 and TOP-008, were effective anti-cancer agents when tested in vitro and in vivo against human breast and prostate tumors. Our studies also indicated that TOP-008 induced the initiation of the caspase cascade leading to apoptosis. Previous toxicology studies in rodents and dogs, as well as a Phase I study in humans, showed NCO-700 to be a well-tolerated, non-toxic compound. Taken together with our current findings, these results suggest that this class of compounds has the potential to be relatively safe, new chemotherapeutic agents for refractory breast and prostate cancers.

Analysis of the electrophysiologic effects of short-term oxybutynin on guinea pig and rabbit ventricular cells.

The objective of this study was to investigate the cardioactive properties of oxybutynin, a drug that is widely prescribed for management of voiding dysfunction. Membrane currents were recorded from whole-cell-configured guinea pig ventricular myocytes, and action potentials were recorded from guinea pig and rabbit papillary muscles. L-type Ca2+ current (I(Ca),L), inward-rectifier K+ current (I(K1)), and delayed-rectifier K+ current (I(K)) were unaffected by < or = 1 microM oxybutynin, and inhibited by higher concentrations. The concentrations that reduced the currents to one-half of predrug control amplitude (K0.5) were as follows: 1(Ca),L, 16.1 microM, I(K1), 18.2 microM, rapidly activating I(K)(I(Kr)), 11.4 microM, and slowly activating I(K)(I(Ks)), 28.7 microM. Action-potential durations at 20 and 90% repolarization (APD20, APD90) were unaffected by oxybutynin < or =3 microM in guinea pig papillary muscles driven at 1 Hz; higher concentrations selectively shortened the APD20 by as much as 25% (100 microM), and caused moderate reductions in maximal upstroke velocity. Changes in the action potentials of rabbit papillary muscles were even smaller than in the guinea pig muscles. Because the peak therapeutic plasma concentration of oxybutynin is in the 0.01-0.1 microM range, the results suggest that the drug is highly unlikely to have adverse effects on cardiac electrical activity.

Methionine Adenosyltransferase I/III Deficiency: Novel Mutationsand Clinical Variations

Methionine adenosyltransferase (MAT) I/III deficiency, caused by mutations in the MAT1A gene, is characterized by persistent hypermethioninemia without elevated homocysteine or tyrosine. Clinical manifestations are variable and poorly understood, although a number of individuals with homozygous null mutations in MAT1A have neurological problems, including brain demyelination. We analyzed MAT1A in seven hypermethioninemic individuals, to provide insight into the relationship between genotype and phenotype. We identified six novel mutations and demonstrated that mutations resulting in high plasma methionines may signal clinical difficulties. Two patients-a compound heterozygote for truncating and severely inactivating missense mutations and a homozygote for an aberrant splicing MAT1A mutation-have plasma methionine in the 1,226-1,870 microM range (normal 5-35 microM) and manifest abnormalities of the brain gray matter or signs of brain demyelination. Another compound heterozygote for truncating and inactivating missense mutations has 770-1,240 microM plasma methionine and mild cognitive impairment. Four individuals carrying either two inactivating missense mutations or the single-allelic R264H mutation have 105-467 microM plasma methionine and are clinically unaffected. Our data underscore the necessity of further studies to firmly establish the relationship between genotypes in MAT I/III deficiency and clinical phenotypes, to elucidate the molecular bases of variability in manifestations of MAT1A mutations.

Dissecting the Interactions between NTF2, RanGDP, and the Nucleoporin XFXFG Repeats

We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP. These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran.

A Model for Escherichia coli DNA Polymerase III Holoenzyme Assembly at Primer/Template Ends: DNA TRIGGERS A CHANGE IN BINDING SPECIFICITY OF THE γ COMPLEX CLAMP LOADER

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.

Design, Synthesis, and Biological Evaluation of Symmetrically and Unsymmetrically Substituted Methoctramine-Related Polyamines as Muscular Nicotinic Receptor Noncompetitive Antagonists

The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) muscarinic acetylcholine receptors. All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M(2) and M(3) receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7-25 microM range while not showing any antagonism for muscarinic receptors up to 10 microM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M(2) receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M(2) receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the receptor.

OB-BP1/Siglec-6: A LEPTIN- AND SIALIC ACID-BINDING PROTEIN OF THE IMMUNOGLOBULIN SUPERFAMILY

We report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1 versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K(d) 91 nM), the other two showed weak binding with K(d) values in the 1-2 microM range. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acalpha2-6GalNAcalpha (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels.

Capillary electrophoresis with end-capillary potentiometric detection using a copper electrode.

Potentiometric end-capillary detection in capillary electrophoresis has the advantage of relatively easy miniaturisation without having to compromise the concentration sensitivity. Potentiometric end-capillary detection using a copper electrode is also attractive because of the sensitive detection of many inorganic and organic UV-transparent ions and the ability to work in both direct and indirect mode. In this work, detection of a number of common anions in a tartrate electrolyte at pH 3 was studied. The influence of the end-capillary detection geometry on the detection performance was investigated. An end-capillary detection cell allowing the separation capillary to be changed without the need to realign the detection electrode was constructed and fitted into a commercial CE apparatus. Under the optimal configuration, which was a 25 microm diameter copper electrode aligned coaxially with a 25 microm capillary and positioned at a distance of about 25 microm from the capillary end, excellent peak shapes were achieved and comparison with simultaneous on-capillary photometric detection showed no additional peak broadening. Good sensitivity was obtained, resulting in concentration limits of detection (LODs) in the low microM range and mass LODs in the low amol range. Examples of separations of inorganic and organic anions are presented and the analytical potential of the detection method is assessed.

Irradiation of Amelanotic Melanoma Cells with 532 nm High Peak Power Pulsed Laser Radiation in the Presence of the Photothermal Sensitizer Cu(II)‐Hematoporphyrin: A New Approach to Cell Photoinactivation

Cu(II)-hematoporphyrin (CuHp) was efficiently accumulated by B78H1 amelanotic melanoma cells upon incubation with porphyrin concentrations up to 52 microM. When the cells incubated for 18 h with 13 microM CuHp were irradiated with 532 nm light from a Q-switched Nd: YAG laser operated in a pulsed mode (10 ns pulses, 10 Hz) a significant decrease in cell survival was observed. The cell photoinactivation was not the consequence of a photodynamic process, as CuHp gave no detectable triplet signal upon laser flash photolysis excitation and no decrease in cell survival was observed upon continuous wave irradiation. Thus, it is likely that CuHp sensitization takes place by photothermal pathways. The efficiency of the photoprocess was modulated by different parameters; thus, while varying the amount of added CuHp in the 3.25-26 microM range had little effect, pulse energies larger than 50 mJ and irradiation times of at least 120 s were necessary to induce a cell inactivation of about 50%. The porphyrin-cell incubation time prior to irradiation had a major influence on cell survival, suggesting that the nature of the CuHp microenvironment can control the efficiency of photothermal sensitization.

Antimuscarinic, calcium channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in the circular muscle of guinea-pig colon.

We have analyzed, by the sucrose gap method, the action of otilonium bromide, a quaternary ammonium derivative in use for the symptomatic therapy of irritable bowel syndrome, on the electrical and mechanical responses initiated by different stimuli in the circular muscle of the guinea-pig proximal colon. Otilonium bromide produced a concentration-dependent inhibition of membrane depolarization (IC50 4.1 microM), action potentials (APs) and contraction (IC50 3.7 microM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC50 31 microM) produced by KCl (30 mM), and had a biphasic effect on the cholinergic excitatory junction potential (e.j.p.) produced by single pulse electrical field stimulation: at low concentrations (0.1-0.3 microM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC50 22 microM and 16 microM toward depolarization and contraction), produced a concentration-dependent inhibition. Otilonium bromide eliminated the APs superimposed on the depolarization induced by the tachykinin NK1 receptor agonist, [Sar9]substance P-sulphone and suppressed the corresponding contraction (IC50 43 microM) but had little effect on the sustained membrane depolarization induced by this agonist. On the other hand, otilonium bromide produced a similar inhibitory effect on both membrane depolarization and contraction (IC50 38 microM and 45 microM, respectively) induced by the tachykinin NK2 receptor agonist [betaAla8]neurokinin A (4-10). When tested in the presence of nifedipine (1 microM), otilonium bromide had no effect on the membrane depolarization induced by [Sar9]substance P-sulphone but inhibited in a concentration-dependent manner the depolarization induced by [betaAla8]neurokinin A (4-10) (IC50 41 microM). In contrast, the blocker of receptor-operated cation channels, SKF 96365, inhibited with similar potency the depolarization induced by both [Sar9]substance P-sulphone and [betaAla8]neurokinin A (4-10) (IC50 60 microM and 54 microM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([125I]neurokinin A, Ki 7.2 microM) and an antagonist ([3H]SR 48968, Ki 2.2 microM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK2 receptor. In conclusion, the present findings demonstrate that, in the microM range of concentrations, otilonium bromide acts as a muscarinic and tachykinin NK2 receptor antagonist and as a calcium channel blocker. The latter property is likely to account for its ability to suppress contraction initiated by the tachykinin NK1 receptor agonist. Therefore multiple mechanisms of action account for the ability of otilonium bromide to reduce stimulated motility of intestinal smooth muscle.

Phytotoxins from the leaves of Laggera decurrens.

Upon biological screening of a series of African medicinal plants, substantial phytotoxic activity was found in the leaves of Laggera decurrens (Vahl.) Hepper & Wood (Asteraceae), using a Lemna minor bioassay. Bioassay-guided fractionation of the leaves led to the isolation of two physiologically active compounds: 3-hydroxythymoquinone and 5-acetoxy-2-hydroxythymol, causing death of Lemna minor in the 25-100 microM range. Symptoms were a rapidly developing chlorosis, followed by necrosis of fronds. The compounds also inhibited growth and germination of the grass weed Agrostis capillaris down to 250 microM. The mode of action of both compounds could not be elucidated, but they do not appear to be photosystem II inhibitors.

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes).

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5'-triphosphate. Adenosine, AMP, alpha, beta-methylene adenosine-5'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.

2'Halo-ATP and -GTP analogues: rational phasing tools for protein crystallography.

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2'-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2'-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2'-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2'-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras(Y32W) (fluorescent mutant) and RabS. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of approximately 100 (p21ras) or approximately 10(5) (Rab5), and the resulting equilibrium dissociation constants are in the nm or microM range, respectively. The data demonstrate that 2'halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10(5)-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the microM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.

Alpha actinin-CapZ, an anchoring complex for thin filaments in Z-line.

CapZ is a widely distributed and highly conserved, heterodimeric protein, that nucleates actin polymerization and binds to the barbed ends of actin filaments, preventing the addition or loss of actin monomers. CapZ interaction with actin filaments was shown to be of high affinity and decreased in the presence of PIP2. CapZ was located in nascent Z-lines during skeletal muscle myofibrillogenesis before the striated appearance of thin filaments in sarcomers. In this study, the stabilization and the anchorage of thin filaments were explored through identification of CapZ partners in the Z-line. Fish (sea bass) striated white muscle and its related Z-line proteins were selected since they correspond to the simplest Z-line organization. We report here the interaction between purified CapZ and alpha-actinin, a major component of Z filaments and polar links in Z-discs. Affinity of CapZ for alpha-actinin, estimated by fluorescence and immunochemical assays, is in the microM range. This association was found to be independent of actin and shown to be weakened in the presence of phosphoinositides. Binding contacts on the alpha-actinin molecule lie in the 55 kDa repetitive domain. A model including CapZ/alpha-actinin/titin/actin interactions is proposed considering Luther's 3D Z-line reconstruction.

Vinpocetine selectively inhibits neurotransmitter release triggered by sodium channel activation.

The effects of vinpocetine on internal Na+ (Na(i)), cAMP accumulation, internal Ca2+ (Ca(i)) and excitatory amino acid neurotransmitters release, under resting and under depolarized conditions, was investigated in rat striatum synaptosomes. Veratridine (20 microM) or high K+ (30 mM) were used as depolarizing agents. Results show that vinpocetine in the low microM range inhibits the elevation of Na(i), the elevation of Ca(i) and the release of glutamate and aspartate induced by veratridine depolarization. In contrast, vinpocetine fails to inhibit the rise of Ca(i) and the neurotransmitter release induced by high K+, which are both TTX insensitive responses. Results also show that the inhibition exerted by vinpocetine on all the above veratridine-induced responses is not reflected in PDE activity. Our interpretation of these results is that vinpocetine inhibits neurotransmitter release triggered by veratridine activation of voltage sensitive Na+ channels, but not that triggered by a direct activation of VSCC. Thus, the main mechanism involved in the neuroprotective action of vinpocetine in the CNS is unlikely to be due to a direct inhibition of Ca2+ channels or PDE enzymes, but rather the inhibition of presynaptic Na+ channel-activation unchained responses.