The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Polyamines stabilize left-handed Z-DNA: using X-ray crystallographic analysis, we have found a new type of polyamine (PA) that stabilizes left-handed Z-DNA

There are many great reports of polyamine stabilization of the Z-DNA by bridge conformation between neighboring, symmetry-related Z-DNA in the packing of crystals. However, polyamine binding to the minor groove of Z-DNA and stabilizing the Z-DNA structure has been rarely reported. We proved that the synthesized polyamines bind to the minor groove of Z-DNA and stabilize the conformation under various conditions, by X-ray crystallographic study. These polyamines consist of a polyamine nano wire structure. The modes of the polyamine interaction were changed under different conditions. It is the first example that the crystals consisted of metal free structure. This finding provides a basis for clarifying B-Z transition mechanics.     

Reversal of multidrug resistance by 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide through the reversible inhibition of P-glycoprotein

Overexpression of P-glycoprotein (P-gp) is one of the major obstacles to successful cancer chemotherapy. In this study, we examined the ability of 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide (C-4) to reverse multidrug resistance (MDR) in P-gp expressing KBV20C cells. Treatment of KBV20C cells with C-4 led to a dramatic increase in paclitaxel- or vincristine-induced cytotoxicity without any cytotoxicity by itself. In parallel, C-4 treatment caused an increase in apoptotic cell death by paclitaxel or vincristine. Furthermore, C-4 treatment significantly increases in intracellular accumulation of fluorescent P-gp substrate rhodamine 123, indicating that C-4 treatment leads to reversal of the MDR phenotype resulting from an increased accumulation of anticancer drugs by inhibiting drug efflux function of P-gp. This notion is further supported by the observation that C-4 treatment potentiates paclitaxel-induced G(2)/M arrest of the cell cycle. In addition, the drug efflux function of P-gp was reversibly inhibited by C-4 treatment, while the expression level of P-gp was not affected. Collectively, our results describe the potential of C-4 to reverse the P-gp-mediated MDR phenotype through reversible inhibition of P-gp function, which may make it an attractive new agent for the chemosensitization of cancer cells.     

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.     

Synthesis, characterization and studies on DNA-binding of a new Cu(II) complex with N1,N8-bis(l-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine

A new Cu(II) complex of CuLCl(2) (here, L=N(1),N(8)-bis(1-methyl-4-nitropyrrole-2- carbonyl)triethylenetetramine) had been synthesized and characterized. The structure of the complex was investigated with density functional theory (DFT) calculations. DNA-binding of the Cu(II) complex and its effects on tumor cell viability were firstly studied. The interactions between the complex and calf thymus DNA had been investigated using UV spectra, fluorescent spectra, viscosity and CV (cyclic voltammetry). The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is classical intercalation and the complex can cleave pBR322 DNA. The effects of the CuL on cell viability were tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye assay and the results indicate that the CuL had certain effect on cancer cells.     

Crystal structure of the RNA demethylase ALKBH5 from zebrafish

ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N⁶-methyladenosine (m⁶A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65 Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m⁶A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m⁶A demethylation.     

para- and ortho-Pyridinium aldoximes in reaction with acetylthiocholine

In the oximolysis reaction para-aldoximes K027 and TMB-4 react faster with ATCh than ortho-aldoximes HI-6 and K033. The reaction rate constants at 25 degrees C were 22 M(-1) min(-1) for HI-6 and K033, 230 M(-1) min(-1) for TMB-4 and 306 M(-1) min(-1) for K027. Semi-empirical calculations showed that differences in rates do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance of the oxime group within the molecule. Thermodynamic parameters, DeltaG#, DeltaH# and DeltaS#, were also determined for oximolysis reaction.     

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.     

Different coordination modes of 5,5-diethylbarbiturate in the copper(II) complexes with some aliphatic amines: Synthesis, spectroscopic, thermal and structural studies

Three new copper(II) complexes of 5,5-diethlybarbiturate (barb), [Cu(barb)₂(dmen)]·0.5H₂O (dmen = N,N-dimethylethylenediamine) 1, [Cu(barb)₂(bapa)] (bapa = bis(3-aminopropyl)amine) 2, and [Cu(barb)(apen)](barb)·2H₂O (apen = N,N′-bis(3-aminopropyl)ethylenediamine) 3, have been synthesized and characterized by chemical, spectroscopic and thermal methods. Single crystal X-ray diffraction studies revealed that all complexes are mononuclear. The copper(II) ion exhibits a square-pyramidal coordination geometry in 1 and 3, but a trigonal-bipyramidal geometry in 2. The barb ligand shows different coordination modes. 1 presents the unequal coordination of the barb ligands: one is monodentate (N) and the other one is bidentate (N, O). In 2, both barb ligands are N-coordinated, whereas in 3, one barb ligand is N-coordinated, while the second barb ligand behaves as a counter-ion. The dmen, bapa and apen ligands act as bi-, tri- and tetradentate ligands, respectively. All complexes display a hydrogen-bonded network structure. The IR spectroscopic analysis shows that the ν(CO) stretching frequencies do not correlate predictably with the coordination mode of the barb ligand in 1. Thermal analysis data for 1-3 are in agreement with the crystal structures.     

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Crystal structure and nucleotide binding of the Thermus thermophilus RNA helicase Hera N-terminal domain

DEAD box RNA helicases use the energy of ATP hydrolysis to unwind double-stranded RNA regions or to disrupt RNA/protein complexes. A minimal RNA helicase comprises nine conserved motifs distributed over two RecA-like domains. The N-terminal domain contains all motifs involved in nucleotide binding, namely the Q-motif, the DEAD box, and the P-loop, as well as the SAT motif, which has been implicated in the coordination of ATP hydrolysis and RNA unwinding. We present here the crystal structure of the N-terminal domain of the Thermus thermophilus RNA helicase Hera in complex with adenosine monophosphate (AMP). Upon binding of AMP the P-loop adopts a partially collapsed or half-open conformation that is still connected to the DEAD box motif, and the DEAD box in turn is linked to the SAT motif via hydrogen bonds. This network of interactions communicates changes in the P-loop conformation to distant parts of the helicase. The affinity of AMP is comparable to that of ADP and ATP, substantiating that the binding energy from additional phosphate moieties is directly converted into conformational changes of the entire helicase. Importantly, the N-terminal Hera domain forms a dimer in the crystal similar to that seen in another thermophilic prokaryote. It is possible that this mode of dimerization represents the prototypic architecture in RNA helicases of thermophilic origin.     

ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ,? we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

The beneficial role of different auxins and polyamines at successive stages of somatic embryo formation and development of Panax ginseng in vitro

The production of viable plantlets via somatic embryogenesis in Panax ginseng requires different culture media corresponding to successive developmental stages. The effects of several auxins and polyamines have been tested at various steps. Multiplication of the embryogenic root-derived callus has been optimized on half-strength MS medium supplemented with 3-(benzo[b]selenyl)acetic acid (BSAA, a synthetic auxin) and kinetin; exogenously applied polyamines were deleterious at this stage, causing browning of the callus, diminished capacity of embryo initiation, and an increased tendency to hyperhydricity. BSAA again appeared to be the most favourable auxin at the initiation stage, but here its action was reinforced by the presence of polyamines, spermidine being the most efficient. Among the auxin needed at the next step, i.e., for the regeneration of embryos, the two seleniated auxins BSAA and seleniated 2,4-dichlorophenoxyacetic acid (2,4-D-Se) were the more efficient. For the harmonious development of plantlets, i.e., the simultaneous outgrowth of shoots and roots, the polyamines were favourable, with a greater efficiency for spermine.     

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.     

Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components

In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S + 5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼ 60% and ∼ 35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a ‘looser’ structure promoted by elevated helical mispairing and a high degree of fragmentation.     

Role of mitogen-activated protein kinases in aryl hydrocarbon receptor signaling

Human populations are increasingly exposed to a number of environmental pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls and dioxins. These compounds are activators of the aryl hydrocarbon receptor (AhR) that controls the expression of many genes including those for detoxification enzymes. The regulatory mechanisms of AhR are multi-factorial and include phosphorylation by various protein kinases. Significant progress in the research of mitogen-activated protein kinases (MAPKs) has been achieved in the last decade. Isolated reports have been published on the role of MAPKs in AhR functions and vice versa, with activation of MAPKs by AhR ligands. This mini-review summarizes current knowledge on the mutual interactions between MAPKs and AhR. The majority of studies has been done on cancer-derived cell lines that have impaired cell cycle regulation and lacks the complete detoxification apparatus. We emphasize the importance of the future studies that should be done on non-transformed cells to distinguish the role of MAPKs in cancer and normal cells. Primary cultures of human or rodent hepatocytes that are equipped with a fully functional biotransformation battery or xenobiotics-metabolizing extra-hepatic tissues should be the models of choice, as the results in our experiments confirm.     

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the “firefly squid”, Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189–197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg²⁺, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6–2 µm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 °C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-γ-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.     

Molecular associations and surface-active properties of short- and long-N-acyl chain ceramides

The behaviour of N-hexadecanoylsphingosine (Cer16), N-hexanoylsphingosine (Cer6) and N-acetylsphingosine (Cer2) in aqueous media and in lipid-water systems, monolayers and bilayers has been comparatively examined using Langmuir balance and fluorescence techniques. Cer16 behaves as an insoluble non-swelling amphiphile, not partitioning into the air-water interface, thus not modifying the surface pressure of the aqueous solutions into which it is included. By contrast both Cer6 and Cer2 behave as soluble amphiphiles, up to approx. 100 μM. At low concentrations, they become oriented at the air-water interface, increasing surface pressure in a dose-dependent way up to ca. 5 μM bulk concentration. At higher concentrations, the excess ceramide forms micelles, critical micellar concentrations of both Cer6 and Cer2 being in the 5-6 μM range. When the air-water interface is occupied by a phospholipid, 6Cer2 and Cer6 become inserted in the phospholipid monolayer, causing a further increase in surface pressure. This increase is dose dependent, and reaches a plateau at ca. 2 μM ceramide bulk concentration. Both Cer2 and Cer6 become inserted in phospholipid monolayers with initial surface pressures of up to 43 and 46 mN m⁻¹, respectively, which ensures their capacity to become inserted into cell membranes whose monolayers are estimated to support a surface pressure of about 30 mN m⁻¹. Both Cer2 and Cer6, but not Cer16, had detergent-like properties, such as giving rise to phospholipid-ceramide mixed micelles, when added to phospholipid monolayers or bilayers. The short-chain ceramides form large aggregates and precipitate at concentrations above approx. 100 μM. These results are relevant in cell physiology studies in which short- and long-chain ceramides are sometimes used as equivalent molecules, in spite of their different biophysical behaviour.