Programmed cell death of secretory cavity cells of citrus fruits is associated with Ca2+ accumulation in the nucleus

Key message

An increase in Ca ²⁺ concentration in the nucleus may activate the PCD of secretory cavity cells, and further Ca ²⁺ accumulation contributes to the regulation of nuclear DNA degradation.

Abstract

Calcium plays an important role in plant programmed cell death (PCD). Previously, we confirmed that PCD was involved in the degradation of secretory cavity cells in Citrus sinensis (L.) Osbeck fruits. To further explore the function of calcium in the PCD of secretory cavity cells, we used potassium pyroantimonate precipitation to detect and locate calcium dynamics. At the precursor cell stage of the secretory cavity, Ca²⁺ was only distributed in the cell walls. At the early stage of secretory cavity initial cells, Ca²⁺ in the cell walls was gradually transported into the cytoplasm via pinocytotic vesicles. Although a small amount of Ca²⁺ was present in the nucleus, the TUNEL signal was scarcely observed. At the middle stage of initial cells, a large number of pinocytotic vesicles were transferred to the nucleus, where the vesicle membrane fused with the nuclear membrane to release calcium into the nucleoplasm. In addition, abundant Ca²⁺ aggregated in the condensed chromatin and nucleolus, where the TUNEL signal appeared the strongest. At the late stage of initial cells, the chromatin and nucleolus gradually degraded and disappeared, and the nucleus appeared broken-like, as Ca²⁺ in the cell wall had nearly completely disappeared, and Ca²⁺ in the nucleus was also rapidly reduced. Furthermore, the TUNEL signal also disappeared. These phenomena indicated that an increase in Ca²⁺ concentration in the nucleus might activate the PCD of secretory cavity cells, and further Ca²⁺ accumulation contributed to the regulation of nuclear DNA degradation.     

Protocol for the BAG-RECALL clinical trial: a prospective, multi-center, randomized, controlled trial to determine whether a bispectral index-guided protocol is superior to an anesthesia gas-guided protocol in reducing intraoperative awareness with explicit recall in high risk surgical patients

Background

Sevoflurane has been demonstrated to vasodilate the foeto-placental vasculature. We aimed to determine the contribution of modulation of potassium and calcium channel function to the vasodilatory effect of sevoflurane in isolated human chorionic plate arterial rings.

Methods

Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 and 2 examined the role of the K⁺ channel in sevoflurane-mediated vasodilation. Separate experiments examined whether tetraethylammonium, which blocks large conductance calcium activated K⁺ (K_Ca++) channels (Series 1A+B) or glibenclamide, which blocks the ATP sensitive K⁺ (K_ATP) channel (Series 2), modulated sevoflurane-mediated vasodilation. Series 3 – 5 examined the role of the Ca⁺⁺ channel in sevoflurane induced vasodilation. Separate experiments examined whether verapamil, which blocks the sarcolemmal voltage-operated Ca⁺⁺ channel (Series 3), SK&F 96365 an inhibitor of sarcolemmal voltage-independent Ca⁺⁺ channels (Series 4A+B), or ryanodine an inhibitor of the sarcoplasmic reticulum Ca⁺⁺ channel (Series 5A+B), modulated sevoflurane-mediated vasodilation.

Results

Sevoflurane produced dose dependent vasodilatation of chorionic plate arterial rings in all studies. Prior blockade of the K_Ca++ and K_ATP channels augmented the vasodilator effects of sevoflurane. Furthermore, exposure of rings to sevoflurane in advance of TEA occluded the effects of TEA. Taken together, these findings suggest that sevoflurane blocks K⁺ channels. Blockade of the voltage-operated Ca⁺⁺channels inhibited the vasodilator effects of sevoflurane. In contrast, blockade of the voltage-independent and sarcoplasmic reticulum Ca⁺⁺channels did not alter sevoflurane vasodilation.

Conclusion

Sevoflurane appears to block chorionic arterial K_Ca++ and K_ATP channels. Sevoflurane also blocks voltage-operated calcium channels, and exerts a net vasodilatory effect in the in vitro foeto-placental circulation.     

A flavonoid, 5-hydroxy-3,7-dimethoxyflavone,from Kaempferia parviflora Wall. Ex. Baker as an inhibitor of Ca2+ signal-mediated cell-cycle regulation in yeast

Calcium (Ca²⁺) signal transduction pathways play important roles in the regulation of diverse biological processes in eukaryotes ranging from unicellular (e.g., yeasts) to complex multicellular (e.g., humans) organisms. Small-molecule inhibitors of Ca²⁺-signaling pathways in humans can be of great medical importance, as represented by the immunosuppressants FK506 and cyclosporine A. A high-throughput drug screening assay for inhibitors of Ca²⁺-signaling has been developed on the basis of the ability of test compounds to restore the severe growth defect of a Ca²⁺-sensitive zds1 null-mutant strain YNS17 of Saccharomyces cerevisiae in a medium containing a high concentration of calcium ions. A previous screening of Thai medicinal plants using this yeast-based assay indicated that the crude extract of Kaempferia parviflora Wall. Ex. Baker contains a potent inhibitory activity. The aim of this study was to isolate and characterize the pure compound(s) responsible for this inhibitory activity against Ca²⁺-mediated cell-cycle regulation in yeast. Dichloromethane and methanol extracts of K. parviflora rhizomes were subjected to bioassay-mediated chromatographic fractionation using this yeast [YNS17 (Δzds1) strain]-based assay to screen for and select positive fractions. From the dichloromethane extract, four known flavonoid compounds with significant inhibitory bioactivity were obtained: compounds 1 (5-hydroxy-3,7-dimethoxyflavone), 2 (5-hydroxy-7-methoxyflavone), 3 (5-hydroxy-3,7,4’-trimethoxyflavone) and 4 (5,7-dimethoxyflavone). The inhibitory activity of all four compounds was dose-dependent. Compound 1 exhibited the highest activity and with no observed cytotoxic activity against the yeast. The Ca²⁺ induced severe growth defect, abnormal budding morphology, and G2 cell-cycle delay of the Δzds1 yeast strain were all alleviated or abrogated by 200 μM compound 1. Therefore, we conclude that 5-hydroxy-3,7-dimethoxyflavone possesses a potent inhibitory activity against the Ca²⁺-mediated cell-cycle regulation.     

Oxidation and reduction of sulfite contribute to susceptibility and detoxification of SO2 in Populus × canescens leaves

Key Message

The critical level for SO ₂ susceptibility of Populus × canescens is approximately 1.2 μL L ^?1 SO ₂ . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO ₂ detoxification.

Abstract

In the present study, uptake, susceptibility and metabolism of SO₂ were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO₂ detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO₂ (0.65, 0.8, 1.0, 1.2 μL L^?1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO₂ assimilation, SO₂ uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO₂ that confers injury to the leaves was 1.2 μL L^?1 SO₂. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO₂ uptake of the plant shoot, whereas SO₂ uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO₂ revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.     

Steroidal aromatase inhibitors inhibit growth of hormone-dependent breast cancer cells by inducing cell cycle arrest and apoptosis

Different hormonal therapies are used for estrogen receptor positive (ER⁺) breast cancers, being the third-generation of aromatase inhibitors (AIs), an effective alternative to the classical tamoxifen. AIs inhibit the enzyme aromatase, which is responsible for catalyzing the conversion of androgens to estrogens. In this study, it was evaluated the effects of several steroidal AIs, namely 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), on cell proliferation, cell cycle progression and cell death in an ER⁺ aromatase-overexpressing human breast cancer cell line (MCF-7aro). All AIs induced a decrease in cell proliferation and these anti-proliferative effects were due to a disruption in cell cycle progression and cell death, by apoptosis. AIs 1 and 16 caused cell cycle arrest in G₀/G₁, while AIs 12 and 13a induced an arrest in G₂/M. Moreover, it was observed that these AIs induced apoptosis by different pathways, since AIs 1, 12 and 13a activated the apoptotic mitochondrial pathway, while AI 16 induced apoptosis through activation of caspase-8. These results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer cells and will also highlight the importance of AIs as inducers of apoptosis in hormone-dependent breast cancers.     

Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response

Key message

Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death.

Abstract

Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants.     

Mechanisms of thaxtomin A-induced root toxicity revealed by a thaxtomin A sensitive <Emphasis Type="Italic">Arabidopsis</Emphasis> mutant (<Emphasis Type="Italic">ucu2</Emphasis>-<Emphasis Type="Italic">2/gi</Emphasis>-<Emphasis Type="Italic">2</Emphasis>)

Key message

The Arabidopsis mutant ( ucu2 - 2/gi - 2 ) is thaxtomin A, isoxaben and NPA-sensitive indicated by root growth and ion flux responses providing new insights into these compounds mode of action and interactions.

Abstract

Thaxtomin A (TA) is a cellulose biosynthetic inhibitor (CBI) that promotes plant cell hypertrophy and cell death. Electrophysiological analysis of steady-state K⁺ and Ca²⁺ fluxes in Arabidopsis thaliana roots pretreated with TA for 24 h indicated a disturbance in the regulation of ion movement across the plant cell membrane. The observed inability to control solute movement, recorded in rapidly growing meristematic and elongation root zones, may partly explain typical root toxicity responses to TA treatment. Of note, the TA-sensitive mutant (ucu2-2/gi-2) was more susceptible with K⁺ and Ca²⁺ fluxes altered between 1.3 and eightfold compared to the wild-type control where fluxes altered between 1.2 and threefold. Root growth inhibition assays showed that the ucu2-2/gi-2 mutant had an increased sensitivity to the auxin 2,4-D, but not IAA or NAA; it also had increased sensitivity to the auxin efflux transport inhibitor, 1-naphthylphthalamic acid (NPA), but not 2,3,5- Triiodobenzoic acid (TIBA), when compared to the WT. The NPA sensitivity data were supported by electrophysiological analysis of H⁺ fluxes in the mature (but not elongation) root zone. Increased sensitivity to the CBI, isoxaben (IXB), but not dichlobenil was recorded. Increased sensitivity to both TA and IXB corresponded with higher levels of accumulation of these toxins in the root tissue, compared to the WT. Further root growth inhibition assays showed no altered sensitivity of ucu2-2/gi-2 to two other plant pathogen toxins, alternariol and fusaric acid. Identification of a TA-sensitive Arabidopsis mutant provides further insight into how this CBI toxin interacts with plant cells.

     

Di- and polynuclear silver(I) saccharinate complexes of tertiary diphosphane ligands: synthesis, structures, in vitro DNA binding, and antibacterial and anticancer properties

A series of new silver(I) saccharinate (sac) complexes, [Ag₂(sac)₂(μ-dppm)H₂O]·H₂O (1), {[Ag₂(μ-sac)₂(μ-dppe)]·3H₂O·CH₂Cl₂} _n (2), [Ag₂(μ-sac)₂(μ-dppp)] _n (3), and [Ag(sac)(μ-dppb)] _n (4) [dppm is 1,1-bis(diphenylphosphino)methane, dppe is 1,2-bis(diphenylphosphino)ethane, dppp is 1,3-bis(diphenylphosphino)propane, and dppb is 1,4-bis(diphenylphosphino)butane], have been synthesized and characterized by C, H, N elemental analysis, IR spectroscopy, ¹H NMR, ¹³C NMR, and ³¹P NMR spectroscopy, electrospray ionization mass spectrometry, and thermogravimetry–differential thermal analysis. Single-crystal X-ray studies show that the diphosphanes act as bridging ligands to yield a dinuclear complex (1) and one-dimensional coordination polymers (2 and 4), whereas the sac ligand adopts a μ₂-N/O bridging mode in 2, and is N-coordinated in 1 and 4. The interaction of the silver(I) complexes with fish sperm DNA was investigated using UV–vis spectroscopy, fluorescence spectroscopy, and agarose gel electrophoresis. The binding studies indicate that the silver(I) complexes can interact with fish sperm DNA through intercalation, and complexes 1 and 3 have the highest binding affinity. The gel electrophoresis assay further confirms the binding of the complexes with the pBR322 plasmid DNA. The minimum inhibitory concentrations of the complexes indicate that complex 1 exhibits very high antibacterial activity against standard bacterial strains of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, being much higher than those of AgNO₃, silver sulfadiazine, ciprofloxacin, and gentamicin. Moreover, complexes 1–3 exhibit very high cytotoxic activity against A549 and MCF-7 cancer cell lines, compared with AgNO₃ and cisplatin. The bacterial and cell growth inhibitions of the silver(I) complexes are closely related to their DNA binding affinities.     

Synthesis of 1,6-Diaryl-5,7-dioxo(dithio)imidazo[1,2-a] [1,3,5]triazines. A Novel Pathway for Imidazo[1,2-a] [1,3,5]triazine Systems Obtained via 1-(Imidazolin-2-yl)urea and Thiourea Derivatives

Novel 1,6-diaryl-5,7(1H)dioxo(dithio)-2,3-dihydroimidazo[1,2-a][1, 3, 5]triazines 8, and 9 were synthesized by cyclization of the respective 1-(imidazolin-2-yl)ureas 4 or thioureas 6 with phosgene or thiophosgene in the presence of bases. 1-Aryl-2-aminoimidazolines 1 reacting with arylisocyanates 2 or arylisothiocyanates 3 form a mixture of isomeric imidazolin-2-yl 4 and 6 and imidazolin-3-yl 5 and 7 urea or thiourea derivatives. Isomers 4 and 6 can be easily separated and used for the cyclization reaction. The structures of the main intermediates and the final target compounds were confirmed by ¹H-NMR spectral analysis. Discussion of the possible course of the reactions is also presented.     

Characterization of bio-related vanadium and zinc complexes containing tetradentate dithiolate-disulfide, -diamine and -amine-amide ligands

The reaction of [VCl₃(PMe₂Ph)₃] _{with HSS′S′SH (where the HS are thiophenolate and the S′ thioether functions, respectively), H2–1, yields [VCl(μ-SS′S′S)]2 (3) with one of the thiolate groups of each of the two ligands in the bridging mode. Reaction of Na2–1 with [VOCl2(thf)2] leads to a polymeric product of composition [VO(SS′S′S)]x (4). The products obtained from the reaction between [VOCl2(thf)2] and NaSNNSNa, Na2–2, (S is thiophenolate, N the amine function) depend on subtle changes in the diamine backbone of this ligand: If the amine functions are linked by -CH2CH2– (2a), the tetranuclear V^IV complex [V(SNNS)μ-O]4 (5) is formed alongside the V^III complex [VCl(SNNS)]. If the backbone is -CH(Me)CH(Me)- (2b), [VO(SNNS)] (7) and the dinuclear, asymmetrically oxo-bridged V^IV complex [{(SNN ^– S)(thf)V}μ-O{V(SNN ^– S)}] (8) are obtained. In 8, one amine of each of the two ligands is deprotonated to the amide group. In either case, the complexation is accompanied by oxidation of the thiolates to disulfides, leading to the generation of teraazatetrathio-cycloeicosanes (6a/b). Compounds 5 and 8·2THF have been structurally characterized by X-ray analyses. The connectivities have further been established for 3·2CH2Cl2 and for 6b, which exhibits the same conformation as formally characterized 6a. The cluster compound 5 is stabilized by an extended intramolecular N-H...O and N-H...S) hydrogen-bonding network. In 7·2THF, one of the THFs of crystallization is hydrogen-bonded to the NH of the penta-coordinated {VO(SNN ^– S)} moiety; further, there is an intramolecular hydrogen bond between one of the thiolates of this tetragonal-pyramidal half of the molecule and the NH of the octahedral {VO(SNN ^– S)thf} half. The generation of the ligand 2b from its precursor compound, the zinc complex [Zn(SNNS)] (9) leads to the structural characterization of 9·CH3OH with a large SZnS bite angle and a strong hydrogen bond between the methanolic OH and one of the thiolate sulfurs. The relevance of these compounds in biological systems is discussed.}     

Enhanced β-turn conformational stability of tripeptides containing ΔPhe in cis over trans configuration

Conformations of three pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc-Gly-Δ^Z/EPhe-Phe-p-NA (Z- p -NA and E- p -NA), Boc-Gly-Δ^Z/EPhe-Phe-OMe (Z-OMe and E-OMe), and Boc-Gly-Δ^Z/EPhe-Phe-OH (Z-OH and E-OH) were compared on the basis of CD and NMR studies in MeOH, TFE, and DMSO. The CD results were used as the additional input data for the NMR-based calculations of the detailed solution conformations of the peptides. It was found that Z- p -NA, E- p -NA, Z-OMe, and Z-OH adopt the β-turn conformations and E-OMe and E-OH are unordered. There are two overlapping type III β-turns in Z- p -NA, type II’ β-turn in E- p -NA, and type II β-turn in Z-OMe and Z-OH. The results obtained indicate that in the case of methyl esters and peptides with a free carboxyl group, Δ^ZPhe is a much stronger inducer of ordered conformations than Δ^EPhe. It was also found that temperature coefficients of the amide protons are not reliable indicators of intramolecular hydrogen bonds donors in small peptides.     

Platinum(II) chloride indenyl complexes: electrochemical and biological evaluation

Four platinum(II) complexes of general formula [PtCl(??¹-C₉H₇)L₂] [where L₂ is 1,2-bis(diphenylphosphino)ethane (dppe) 1 or cycloocta-1,5-diene (cod) 3] and [PtCl₂L₂] (where L₂ is dppe 2 or cod 4) were studied. Inhibition growth assays on human tumor cell lines evidenced for 1 and 3 an antiproliferative effect and, interestingly, the cytotoxic effect exerted by 1 is similar to that of cisplatin. Electrochemical and NMR measurements allowed us to determine the structural and redox properties. Investigation of the mechanism of action responsible for the cytotoxicity demonstrated a weak capacity of interacting with DNA. Some experiments performed on rat liver mitochondria indicate that 1 acts as an inducer of the mitochondrial permeability transition, thus leading to the release of proapoptotic factors, such as cytochrome?c and apoptosis-inducing factor.     

Microbial transformation of 20(S)-protopanaxatriol by Absidia corymbifera and their cytotoxic activities against two human prostate cancer cell lines

Seven hydroxylates of 20(S)-protopanaxatriol (1) transformed by Absidia corymbifera AS 3.3387 were isolated and identified by spectral methods including 2D-NMR. Among them, 7β-hydroxyl-20(S)-protopanaxatriol (2), 7α-hydroxyl-20(S)-protopanaxatriol (3), and 7β, 15α-dihydroxyl-20(S)-protopanaxatriol (7) are new compounds. The metabolites 2, 6, 7, and 8 showed the more potent inhibitory effects against DU-145 and PC-3 cell lines than the substrate.     

Two novel hydroperoxylated products of 20(S)-protopanaxadiol produced by Mucor racemosus and their cytotoxic activities against human prostate cancer cells

Microbial transformation of 20(S)-protopanaxadiol (1) by Mucor racemosus AS 3.205 yielded two novel hydroperoxylated metabolites and three known hydroxylated metabolites. The structures of the metabolites were identified as 26-hydroxyl-20(S)-protopanaxadiol (2), 23,24-en-25-hydroxyl-20(S)-protopanaxadiol (3), 25,26-en-24(R)-hydroperoxyl-20(S)-protopanaxadiol (4), 23,24-en-25-hydroperoxyl-20(S)-protopanaxadiol (5), and 25-hydroxyl-20(S)-protopanaxadiol (6). 4 and 5 are new compounds. Metabolites 2, 4, and 5 showed the more potent inhibitory effects against DU-145 and PC-3 cell lines than the substrate.     

Relating normal vibrational modes to local vibrational modes: benzene and naphthalene

Local vibrational modes can be directly derived from normal vibrational modes using the method of Konkoli and Cremer (Int J Quant Chem 67:29, 1998). This implies the calculation of the harmonic force constant matrix F ^q (expressed in internal coordinates q) from the corresponding Cartesian force constant matrix f ^x with the help of the transformation matrix U?=?WB ^?(BWB ^?)^?1 (B: Wilson’s B-matrix). It is proven that the local vibrational modes are independent of the choice of the matrix W. However, the choice W?=?M ^?1 (M: mass matrix) has numerical advantages with regard to the choice W?=?I (I: identity matrix), where the latter is frequently used in spectroscopy. The local vibrational modes can be related to the normal vibrational modes in the form of an adiabatic connection scheme (ACS) after rewriting the Wilson equation with the help of the compliance matrix. The ACSs of benzene and naphthalene based on experimental vibrational frequencies are discussed as nontrivial examples. It is demonstrated that the local-mode stretching force constants provide a quantitative measure for the C–H and C–C bond strength.     

2-Phenylpyridine ruthenacycles as effectors of glucose oxidase activity: inhibition by RuII and activation by RuIII

Cyclometalated Ru^II derivatives of 2-phenylpyridine (Hphpy) [Ru(phpy)(bpy)₂]Cl (1a) and [Ru(phpy)(phen)₂]Cl (1b) (bpy is 2,2′-bipyridine, phen is 1,10-phenanthroline) behave as noncompetitive inhibitors of glucose oxidase from Aspergillus niger in the enzyme-catalyzed oxidation of d-glucose by O₂ into the corresponding lactone at pH 5.0 and 25 °C. The enzymatic activity has been measured by monitoring the O₂ consumption. The inhibition constants K _i are 0.036 and 0.017 M for 1a and 1b, respectively, indicating that 1b inhibits the enzymatic activity more efficiently than 1a. The well-known coordination compound [Ru(bpy)₃]Cl₂ (2) behaves, in contrast, as a competitive inhibitor, with K _i = 0.018 M under the same conditions. The monophasic consumption of O₂ in the case of 1a, 1b, and 2 is replaced by a distinct two-phase kinetics in the presence of the cyclometalated Ru^III compound [Ru(phpy)(bpy)₂]Cl₂ (3), which was obtained from 1a in the presence of a large excess of H₂O₂ and the iron TAML activator. Interestingly, the rates of the first and the second phases are influenced by 3 in a different way. The rate of the first phase is noticeably higher in the presence of Ru^III, although the dependence is nonmonotonic and maximal acceleration is observed at the lowest loadings of 3. The rate of the second phase decreases monotonically on increasing the concentration of the ruthenium complex in solution. The nonmonotonic action of 3 was confirmed by using the doubly cyclometalated Ru^III derivative [Ru(phpy)₂(bpy)]Cl. The diverse rate variations induced by 3 accounted for acceleration by Ru^III of the O₂ reduction by the reduced form of glucose oxidase during the first phase, which ceases after the enzymatic reduction of Ru^III to the Ru^II species, the latter behaving similarly to 1a as the inhibitor of the enzyme.     

Synthesis and Reactions of Some 2-Aminobenzothiazoles

N-(2-Benzothiazolyl)- and N-(6-methoxy-2-benzothiazolyl)cyanoacetamides 4, 5 resulted in the reaction of 2-aminobenzothiazole 1 or its 6-methoxy derivative 2 with 1-cyanoacetyl-3,5-dimethylpyrazole 3. Both cyanoacetylamides 4 and 5 have been transformed into the corresponding 2-oxo-2H-pyrimido[2,1-b]-benzothiazole-3-carbonitrile 8 and its 8-methoxy derivative 9 by reaction with triethyl orthoformate, followed by cyclization.