Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

BackgroundIn photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg^2 +.MethodsTK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography.ResultsCrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg^2 +. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg^2 +-dependent and proceeds with a slow kinetics. The 3D–structure of CrTK without cofactors (CrTK_apo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg^2 + participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation.ConclusionThese evidences indicate that Mg^2 + is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg^2 + is involved in the control of redox sensitivity of CrTK.General significanceThe importance of Mg^2 + in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg^2 + in chloroplasts.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ~4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg²⁺ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg²⁺directly, can nonetheless inhibit Mg²⁺ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg²⁺ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.     

Magnesium uptake of Arabidopsis transporters,AtMRS2-10 and AtMRS2-11, expressed in Escherichia coli mutants: Complementation and growth inhibition by aluminum

Magnesium (Mg^2 +) plays a critical role in many physiological processes. Mg^2 + transport systems in Salmonella have been well documented, but those in Escherichia coli have not been fully elucidated. We examined the effects of corA, mgtA, yhiD and corC gene deletion on Mg^2 + transport in E. coli. We obtained every combination of double, triple and quadruple mutants. The corA and mgtA double mutant required addition of 10 mM Mg^2 + to Luria-Bertani (LB) medium for growth, and the corA, mgtA and yhiD triple mutant TM2 required a higher Mg^2 + concentration. The Mg^2 + requirement of the quadruple mutant was similar to that of TM2. The results demonstrated that either CorA or MgtA is necessary for normal E. coli growth in LB medium and that YhiD plays a role in Mg^2 + transport under high Mg^2 + growth conditions in E. coli. The Arabidopsis Mg^2 + transporters, AtMRS2-10 and AtMRS2-11, were heterologously expressed in TM2 cells. TM2 cells expressing AtMRS2-10 and AtMRS2-11 could grow in LB medium that had been supplemented with 1 mM Mg^2 + and without Mg^2 + supplementation, respectively, and cell growth was inhibited by 2 mM AlCl₃. The results indicated that the growth of TM2 expressing AtMRS2-10 and AtMRS2-11 reflected these AtMRS2 function for Mg^2 + and aluminum. The E. coli TM2 cells are useful for functional analysis of Arabidopsis MRS2 proteins.     

Aldosterone signaling through transient receptor potential melastatin 7 cation channel (TRPM7) and its α-kinase domain

We demonstrated a role for the Mg^2 + transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg^2 + and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg^2 +]_i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg^2 + responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg^2 + influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg^2 +-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.     

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (MstopoI) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage in the absence of Mg²⁺, but religation needs exogenously added Mg²⁺. One molecule of Mg²⁺ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim (topoisomerase-primase) domain in MstopoI comprising the Mg²⁺ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg²⁺ in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg²⁺ dependent for DNA cleavage unlike MstopoI and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg²⁺-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Identification and characterization of a novel endoglucanase (CMCase) isolated from the larval gut of Bombyx mori

While screening for cellulase-producing fungi from insect gut, a fungus with high endoglucanase (carboxymethyl cellulase; CMCase) activity was isolated from the larval gut of Bombyx mori. Based on morphological characteristics and using an 18S rRNA-based molecular phylogenetic approach, the fungus, strain BMC-2, was identified as a Mucor sp. expressing a novel alkalotolerant cellulase. The maximum production of cellulase by the BMC-2 strain was observed at 55 °C and pH 8.0. The CMCase activity was inhibited by Cu^2 + > Na⁺ > Zn^2 + > Mg^2 + > Ba^2 +, and induced by Ca^2 +, Mn^2 +, Fe^2 +, and K⁺.     

Molecular dynamics simulation of cation–phospholipid clustering in phospholipid bilayers: Possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K⁺, Mg^2 +, and Ca^2 + ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca^2 + ions with lipids is significantly stronger than that of K⁺ and Mg^2 + ions, regardless of the composition of the lipid bilayer. The binding of Ca^2 + ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K⁺- and Mg^2 +-containing systems. The Ca^2 + ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength—most notably for Ca^2 +. The formation of cation–lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca^2 +–phospholipid clusters across apposed lipid bilayers can work as a “cation glue” to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

Immunosuppressant FK506 decreases the intracellular magnesium in the human osteoblast cell by inhibiting the ERK1/2 pathway

AimsPrevious studies reported that FK506 influences bone mineralizing and hypomagnesemia, and also has immune modifying properties. This study examined whether or not the function of Mg²⁺ in bone metabolism plays a role in the loss of bone volume caused by immunosuppressants.Main methodsThe effects of the FK506 treatment on the intracellular magnesium and lactate dehydrogenase (LDH) activity were examined in cultured human osteoblasts (HOB) cells. The magnesium concentration was determined using microfluorescence techniques and atomic absorption spectrophotometry. Western blotting was used to measure the level of extracellular signal-regulated kinases 1/2 (ERK 1/2) activation.Key findingsFK506 (0.1 μM) did not affect cell death in HOB cells after a 24 hour treatment but decreased the level of ERK 1/2 activation. In HOB cells, the mean [Mg²⁺]i after exposure to a 1 mM extracellular Mg²⁺ ([Mg²⁺]o) buffer was 0.53 ± 0.01 mM (n = 25). Exposure to 100 nM FK506 produced a significant decrease in [Mg²⁺]i (0.41 ± 0.01 mM). The ERK inhibitor (PD98059) and FK506 produced similar effects but they were not cumulative.SignificanceThis study examined the role of ERK1/2 activation on the regulation of magnesium in HOB. These results suggest that the inhibition of ERK phosphorylation is an essential intermediate in the effects of FK506 on magnesium. Overall, FK506 causes bone disorders partly by decreasing [Mg²⁺]i accompanied by the inhibition of ERK 1/2.     

Calorimetric and spectroscopic investigations of the binding of metallated porphyrins to G-quadruplex DNA

BackgroundThe human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure.MethodsIsothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na⁺] or [K⁺] ≈ 0.15 M).ResultsThe apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a “four-independent-sites model”. The two highest-affinity sites exhibit a K in the range of 10⁸ to 10¹⁰ M^− 1 with the two lower-affinity sites exhibiting a K in the range of 10⁴ to 10⁵ M^− 1. Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a “two-independent-sites model” in which only the end-stacking binding mode is observed with a K in the range of 10⁴ to 10⁵ M^− 1.ConclusionsIn the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an “end stacking” mechanism for the higher affinity binding mode and an “intercalation” mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the “end-stacking” mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands.General significanceThe preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

How do reactive oxygen species and calcium trigger mitochondrial membrane permeabilisation?

BackgroundMitochondrial membrane permeabilisation (MMP) is classically considered as a point of no return in several forms of cell death and is involved in numerous diseases such as cancer, neurodegenerative disorders or ischemia/reperfusion injuries. Many studies established that reactive oxygen species (ROS) and Ca^2 + were the prominent inducers of MMP. However, the mechanisms connecting ROS and Ca^2 + to the players of MMP are still a matter of debate.Scope of reviewThe aim of this review is to summarise the various studies related to the mechanisms of ROS- and Ca^2 +-induced MMP. Several lines of evidence suggest that ROS and Ca^2 + cooperate to induce MMP but the molecular details of the ROS–Ca^2 +-MMP network remain controversial. We then discuss recent data depicting this topic.Major conclusionsCytotoxic stimuli may be transduced within the cell by ROS and Ca^2 + increases. In most models, Ca^2 + and ROS can cooperate to induce MMP. Moreover, several data suggest that MMP increases mitochondrial Ca^2 + and ROS which therefore amplify the cytotoxic signal. Intriguingly, many reports have identified players of MMP as direct ROS targets. On the contrary, direct targets of Ca^2 + remain elusive. At the same time, the mechanisms by which mitochondrial Ca^2 + overload induces ROS generation are well documented. Upon these observations, we hypothesise that Ca^2 + cannot directly induce MMP and requires ROS production as a mandatory step.General significanceGiven the importance of Ca^2 +- and ROS-induced MMP in diseases, we expect that a better understanding of this process will lead to the development of novel therapies.     

Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.     

A rational design strategy of the novel topoisomerase II inhibitors for the synthesis of the 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin with antitumor activity by diminishing the relaxation reaction of topoisomerase II-DNA decatenation

A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis of the esterification and amidation substituted 4′-demethylepipodophyllotoxin (DMEP) derivates was developed in order to discover the potential antitumor prodrug. Firstly, according to the structure–activity relationship, drug combination principle and bioisosterism, the –COO– and the –NH– bond substituents at the 4 position of cycloparaffin would be a great modification direction to improve antitumor activity of 4′-demethylepipodophyllotoxin (DMEP). Secondly, from the prodrug principle view, the esterification and amidation at the C-4 position of DMEP would be two useful structure modifications for improve solubility. Thirdly, from the activity pocket in Topo II-DNA cleavage complex point of view, a series of heterocyclic with pharmacological activity were chosen as module for improving antitumor activity by binding with Topo II. Finally, nine novel esterification and amidation DMEP derivates were designed and synthesized for the potential Topo II inhibitors with the superior biological activity. All the novel compounds exhibited promising in vitro antitumor activity, especially 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin (compound 1). The antitumor activity of compound 1 against tumor cell line HeLa (i.e., the IC₅₀ value of 0.60 ± 0.20 μM), A549 (i.e., the IC₅₀ value of 3.83 ± 0.08 μM), HepG2 (i.e., the IC₅₀ value of 1.21 ± 0.05 μM), and BGC-823 (i.e., the IC₅₀ value of 4.15 ± 1.13 μM) was significantly improved by 66, 16, 12, and 6 times than that of the clinically important podophyllum anticancer drug etoposide (i.e., the IC₅₀ values of 15.32 ± 0.10, 59.38 ± 0.77, 67.25 ± 7.05, and 30.74 ± 5.13 μM), respectively. Compound 1 could arrest HeLa cell cycle G₂/M and induce apoptosis by strongly diminishing the relaxation reaction of Topo II-DNA decatenation. The correctness of rational drug design was strictly demonstrated by the bioactivity test.     

Cholesterol enrichment of rabbit platelets enhances the Ca2 + entry pathway induced by platelet-derived secondary feedback agonists

AimsHypersensitivity of platelets due to increased platelet cholesterol levels has been reported in hypercholesterolemia. However, the signaling pathways linking increased platelet reactivity and cholesterol contents are not fully understood. This study aims to determine the direct effect of cholesterol enrichment of platelets on the pathways including Ca^2 + mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A₂ (TXA₂).Main methodsIn vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca^2 + mobilization was monitored using platelets loaded with fura-PE3/AM, a fluorescent calcium indicator. Released ATP and TXB₂ from platelets were measured by a luciferin–luciferase ATP assay system and a TXB₂ ELISA Kit, respectively.Key findingsCholesterol enrichment of rabbit platelets significantly enhanced Ca^2 + mobilization induced by thrombin, accompanying an augmented Ca^2 + entry. The augmentation of Ca^2 + entry by cholesterol enrichment was significantly suppressed by treatment with inhibitors for secondary feedback agonists. In cholesterol-enriched platelets, the amount of released ATP or TXB₂ induced by thrombin was not significantly altered in comparison with control platelets, whereas an increase in [Ca^2 +]_i induced by ADP or U46619, a TXA₂ mimetic, was significantly enhanced.SignificanceThese results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca^2 + mobilization via ADP/TXA₂-dependent augmentation of the Ca^2 + entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents.