Synthesis,characterization and antitumor activity of a series of N-substituted iminodiacetato(diammine) platinum(II) complexes

A series of water-soluble N-substituted iminodiacetato (diammine)platinum(II) complexes [Pt(NRIDA)(NH₃)₂] have been synthesized and characterized by measurement of physical properties (conductivity and pH) and by various spectroscopic techniques (infrared, ¹H and ¹³C{¹H} nuclear magnetic resonance). The iminodiacetate ligand is coordinated to platinum through an O,N linkage. The results obtained suggest that these complexes are relatively stable for more than 24 h in aqueous solution. Preliminary in vitro and in vivo screening test for antitumor activity of these complexes against L1210 murine leukemia were performed. Many of complexes had acceptable in vitro cytotoxicity, but none displayed a significant level of in vivo antitumor efficacy.     

Effect of an anti-tumor platinum complex,Pt(II)diaminotoluene,on mitochondrial membrane properties

The effects of platinum complexes, selected for their potent anti-tumor activities, have been studied on rat liver mitochondria. Among the mitochondrial properties which have been studied, the most marked effects of platinum complexes were obtained on functions linked to the inner membrane.cis-Pt(II)(3,4-diaminotoluene) dichloride is shown to stimulate state 4 respiration. It inhibits the phosphate transport into mitochondria, decreases the accumulation of Ca²⁺, and induces a more rapid release of the accumulated Ca²⁺. A release of Mg²⁺ from mitochondria incubated in the absence of added divalent cations, and an efflux of divalent cations from mitochondrial membranes are also observed.All these results indicate a profound modification of the permeability of mitochondrial membrane.     

lac Repressor-operator interaction. IX. The binding of lac repressor to operators containing Oc mutations

Representative members of the six classes of operator constitutive (O^c) point mutations, which have been mapped and well characterized in vivo, were crossed into λφ80 lac phages. The phage DNAs containing the O^c mutations were used to measure the affinity of the lac repressor (R) for each O^c operator by determining the half-lives of the different RO^c complexes in vitro. The results provide evidence that: (a) the higher the constitutive level of β-galactosidase in vivo, as the result of an O^c mutation, the lower the affinity of the lac repressor for that O^c operator, with a maximum difference of two orders of magnitude in affinity of the repressor for the highest O^c tested as compared to the wild type O⁺ operator; (b) the six classes of O^c operators appear to be twofold degenerate, in that two members of each class, which were previously distinguished by mapping, have the same affinity for the lac repressor; (c) an inducer and an anti-inducer have the same effect on the RO^c complexes as on the RO⁺ complexes; (d) the relationship between induction ratios in vivo and the binding constant of the repressor for each O^c mutation in vitro does not follow the mass action equation but rather a more complex dependency, which is discussed.These results suggest a functional symmetry in the lac operator.     

Biological activity of ruthenium nitrosyl complexes

Nitric oxide plays an important role in various biological processes, such as neurotransmission, blood pressure control, immunological responses, and antioxidant action. The control of its local concentration, which is crucial for obtaining the desired effect, can be achieved with exogenous NO-carriers. Coordination compounds, in particular ruthenium(III) and (II) amines, are good NO-captors and -deliverers. The chemical and photochemical properties of several ruthenium amine complexes as NO-carriers in vitro and in vivo have been reviewed. These nitrosyl complexes can stimulate mice hippocampus slices, promote the lowering of blood pressure in several in vitro and in vivo models, and control Trypanosoma cruzi and Leishmania major infections, and they are also effective against tumor cells in different models of cancer. These complexes can be activated chemically or photochemically, and the observed biological effects can be attributed to the presence of NO in the compound. Their efficiencies are explained on the basis of the [Ru^IINO⁺]³⁺/[Ru^IINO⁰]²⁺ reduction potential, the specific rate constant for NO liberation from the [RuNO]²⁺ moiety, and the quantum yield of NO release.     

A critical assessment of the use of lipophilic cations as membrane potential probes

A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP⁺) as membrane potential probes in prokaryotes, uekaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials of lipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand in valid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.     

Thermodynamics of Mg and Ca complexation with AMP,ADP, ATP

The thermodynamic parameters (ΔG, ΔH, ΔS) of complexation have been measured by potentiometric and calorimetric titration for formation of ML and MHL (M  Mg²⁺, Ca²⁺; L  AMP²⁻, ADP³⁻, ATP⁴−). The parameters are interpreted to support a model of inner sphere complexation of the metal cations to the phosphate groups with no evidence of metal-ring interaction in the ML complexes. In the MHL complexes, the protonation (of a ring nitrogen) seemingly leads to ‘backfolding’ interaction between the metal and the ring system in addition to the interaction between the metal and the phosphate groups.     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.     

Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study

Phosphorothioate oligodeoxynucleotides (ODNs) have been extensively investigated in vivo and in vitro for antisense control of gene expression. It has been shown that cellular uptake of phosphorothioate ODNs in some in vitro cell systems increases in the presence of divalent cations. In this work, we analyze the conformation of phosphorothioate ODNs and specific changes induced in it by various divalent cations using circular dichroism (CD) spectroscopy. CD data were obtained with several phosphorothioate ODNs in the absence and presence of the divalent cations Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺. All CD spectra indicated stable conformations of the ODNs in solution. The spectra were strongly dependent on ODN sequence and composition. Some ODNs such as T₂₃ and another with ‘random’ distribution of bases showed CD spectra characteristic of B-form DNA. Other ODNs which had at least three consecutive guanines in their sequences exhibited spectra characteristic of parallel G-tetraplexes. CD spectra of antisense ODNs exhibited specific responses to divalent cations. Changes in the conformation were not simply due to ionic strength effects. Mn²⁺ diminished secondary structure in some ODNs. Group II divalent ions stabilized the parallel G-tetraplexes, and Mg²⁺ generally had the weakest stabilizing efficiency. Each sequence/ion combination had a specific response so these effects cannot be generalized. These sequence-dependent, divalent ion-sensitive, and structurally unique solution conformations may be related to ion-mediated ODN uptake.     

Chemical and biological evaluation of 153Sm and 166Ho complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylphosphonic acid monoethylester) (H4 dotp OEt)

The novel methylphosphonic acid monoethylester (H₄dotp^OEt) has been synthesized and characterized and their complexes with Sm(III) and Ho(III) ions were studied. Dissociation constants of the ligand are lower than those of H₄dota. The stability constants of the Ln(III)-H₄dotp^OEt complexes are surprisingly much lower that those of H₄dota (H₄dota = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) probably due to a lower coordination ability of the phosphonate monoester groups. Acid-assisted decomplexation studies have shown that both complexes are less kinetically inert than the H₄dota complexes, but still much more inert than complexes of open-chain ligands. Nevertheless, the synthesis of ¹⁵³Sm and ¹⁶⁶Ho complexes with this ligand led to stable complexes both in vitro and in vivo. A very low binding of these complexes to hydroxyapatite (HA) and calcified tissues was observed confirming the assumption that a fully ionized phosphonate group(s) is necessary for a strong bone affinity. Both complexes show similar behaviour in vivo and, in general, follow the biodistribution trend of the H₄dota complexes with the same metals.     

p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy

p27^Kip1 (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo.     

Formation of a Stable “Active” Complex between Cytochrome <Emphasis Type="Italic">c</Emphasis> and Yeast Peroxidase

THE mechanism of oxidation of cytochrome c in vivo is still unknown¹. One reason for this is that the usual mitochondrial system involved is insoluble and the membrane binding site is ill-defined and heterogeneous². Solubilization of the terminal oxidase led to the discovery^3–5 of complexes between it and cytochrome c, but whether these involved the protein^4,5, lipid⁶, or haem⁷ groups of the oxidase is uncertain. According to one kinetic interpretation⁸ such complexes are abortive and play no part in catalysis. And the occurrence of an oxidase-c complex in situ has not been proved.     

Further studies on the synthesis of RNA in vitro by enzyme--template complexes isolated from induced lambda lysogens

RNA synthesized in vitro by enzyme-template complexes isolated from λ lysogens at early or late times following induction has been shown by competition-hybridization procedures to resemble messenger RNA transcribed in vivo at the same stage of viral development, and to differ from RNA made in vitro by purified Escherichia coli RNA polymerase. It is demonstrated here that RNA synthesis by such complexes involves elongation of chains which have been started in vivo, rather than initiation of new RNA chains in vitro.     

The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin,Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75^NTR, that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α₂-macroglobulin (α₂M) is neurotoxic. Toxicity is due in part to α₂M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α₂M neurotoxicity. First, unexpectedly the α₂M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α₂M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α₂M-proNGF complexes bind p75^NTR and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α₂M regulates proNGF/p75^NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α₂M causes neurodegeneration in a p75^NTR- and proNGF-dependent manner. α₂M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals.     

IgE-Mediated Enhancement of CD4+ T Cell Responses in Mice Requires Antigen Presentation by CD11c+ Cells and Not by B Cells

IgE antibodies, administered to mice together with their specific antigen, enhance antibody and CD4⁺ T cell responses to this antigen. The effect is dependent on the low affinity receptor for IgE, CD23, and the receptor must be expressed on B cells. In vitro, IgE-antigen complexes are endocytosed via CD23 on B cells, which subsequently present the antigen to CD4⁺ T cells. This mechanism has been suggested to explain also IgE-mediated enhancement of immune responses in vivo. We recently found that CD23⁺ B cells capture IgE-antigen complexes in peripheral blood and rapidly transport them to B cell follicles in the spleen. This provides an alternative explanation for the requirement for CD23⁺ B cells. The aim of the present study was to determine whether B-cell mediated antigen presentation of IgE-antigen complexes explains the enhancing effect of IgE on immune responses in vivo. The ability of spleen cells, taken from mice 1–4 h after immunization with IgE-antigen, to present antigen to specific CD4⁺ T cells was analyzed. Antigen presentation was intact when spleens were depleted of CD19⁺ cells (i.e., primarily B cells) but was severely impaired after depletion of CD11c⁺ cells (i.e., primarily dendritic cells). In agreement with this, the ability of IgE to enhance proliferation of CD4⁺ T cells was abolished in CD11c-DTR mice conditionally depleted of CD11c⁺ cells. Finally, the lack of IgE-mediated enhancemen of CD4⁺ T cell responses in CD23^-/- mice could be rescued by transfer of MHC-II-compatible as well as by MHC-II-incompatible CD23⁺ B cells. These findings argue against the idea that IgE-mediated enhancement of specific CD4⁺ T cell responses in vivo is caused by increased antigen presentation by B cells. A model where CD23⁺ B cells act as antigen transporting cells, delivering antigen to CD11c⁺ cells for presentation to T cells is consistent with available experimental data.     

Expression of dual Nucleotides/Cysteinyl‐Leukotrienes Receptor GPR17 in early trafficking of cardiac stromal cells after myocardial infarction

GPR17 is a G_i-coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a population of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1⁺ cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17⁺ cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1⁺/CD31⁻ cell line derived from normal hearts. These experiments showed a migratory function of the receptor by treatment with UDP-glucose and leukotriene LTD4, two GPR17 pharmacological agonists. The GPR17 function was finally assessed in vivo by treating infarcted mice with Cangrelor, a pharmacological receptor antagonist, which, at least in part, inhibited early recruitment of GPR17⁺ and CD45⁺ cells. These findings suggest a regulation of heart-resident mesenchymal cells and blood-borne cellular species recruitment following myocardial infarction, orchestrated by GPR17.     

Effects of Phosphorylatable Short Peptide-Conjugated Chitosan-Mediated IL-1Ra and igf-1 Gene Transfer on Articular Cartilage Defects in Rabbits

Previously, we reported an improvement in the transfection efficiency of the plasmid DNA-chitosan (pDNA/CS) complex by the utilization of phosphorylatable short peptide-conjugated chitosan (^pSP-CS). In this study, we investigated the effects of ^pSP-CS-mediated gene transfection of interleukin-1 receptor antagonist protein (IL-1Ra) combined with insulin-like growth factor-1 (IGF-1) in rabbit chondrocytes and in a rabbit model of cartilage defects. pBudCE4.1-IL-1Ra+igf-1, pBudCE4.1-IL-1Ra and pBudCE4.1-igf-1 were constructed and combined with ^pSP-CS to form pDNA/^pSP-CS complexes. These complexes were transfected into rabbit primary chondrocytes or injected into the joint cavity. Seven weeks after treatment, all rabbits were sacrificed and analyzed. High levels of IL-1Ra and igf-1 expression were detected both in the cell culture supernatant and in the synovial fluid. In vitro, the transgenic complexes caused significant proliferation of chondrocytes, promotion of glycosaminoglycan (GAG) and collagen II synthesis, and inhibition of chondrocyte apoptosis and nitric oxide (NO) synthesis. In vivo, the exogenous genes resulted in increased collagen II synthesis and reduced NO and GAG concentrations in the synovial fluid; histological studies revealed that pDNA/^pSP-CS treatment resulted in varying degrees of hyaline-like cartilage repair and Mankin score decrease. The co-expression of both genes produced greater effects than each single gene alone both in vitro and in vivo. The results suggest that ^pSP-CS is a good candidate for use in gene therapy for the treatment of cartilage defects and that igf-1 and IL-1Ra co-expression produces promising biologic effects on cartilage defects.