Template-directed synthesis on the oligonucleotide d(C7-G-C7)

When the deoxynucleotide template d(C7-G-C7) is incubated with the activated nucleotides 2-MeImpG and 2-MeImpC, a series of oligomers of G up to the sevenmer and a series of copolymers of composition GnC with n = 3 to 13 are formed. Oligomers GnC with n greater than 7 are completely degraded by pancreatic ribonuclease, establishing that they contain a 3' to 5' internucleotide bond between 5'-C and 3'-G within a sequence of the form (pG)ipC(pG)j. As expected, (pG)7-Cp and (pG)6-Cp are major hydrolysis products. Detailed analysis of the product distribution shows that a substantial fraction of the oligomeric products are of the type (pG)ipC(pG)j with i less than 7. This shows that product synthesis does not necessarily begin at the 3' terminus of the template. The significance of this finding in terms of the origin of molecular replication is discussed.     

Architecture of models for prebiotic synthesis of nucleic acids: template-directed synthesis of oligoguanylates using N-cyanoimidazole

N-Cyanoimidazole is an efficient condensing agent for the polymerization of guanosine 5'-phosphate (pG) on a poly(C) template in an aqueous solution. At 0 degree C, up to about 30% of input pG was converted to a mixture of oligomers with a mean chain length of up to 7. The effect of divalent metal ions in the polymerization of pG on a poly(C) template was not so considerable as in that of oligo(A) on a poly(U) template. In the polymerization of pG, the moderate yields were obtaind in the presence of Co2+, Ni2+ and Cu2+.     

cis-Platinum induced distortions in DNA. Conformational analysis of d(GpCpG) and cis-pt(NH3)2[d(GpCpG)], studied by 500-MHz NMR

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G-G chelated deoxytrinucleosidediphosphate platinum complex cis-Pt(NH3)2[d(GpCpG], on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis-Pt(NH3)2[d(Gp)]2, cis-Pt(NH3)2[d(pG)]2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis-Pt(NH3)2]2+. Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring. This feature is also apparent in ribose mononucleotides and is possibly caused by an increased anomeric effect. In cis-Pt(NH3)2[d(pG)]2 the phase angle of pseudorotation of the S-type sugar ring is 20 degrees higher than in 'free' d(pG) which might be an indication for an ionic interaction between the positive platinum and the negatively charged phosphate. It appears that d(GpCpG) reverts from a predominantly random coil to a normal right-handed B-DNA-like single-helical structure at lower temperatures, whereas the conformational features of cis-Pt(NH3)2[d(GpCpG)] are largely temperature-independent. In the latter compound much conformational freedom along the backbone angles is seen. The cytosine protons and deoxyribose protons exhibit almost no shielding effect as should normally be exerted by the guanine bases in stacking positions. This is interpreted in terms of a 'turning away' of the cytosine residue from both chelating guanines. Conformational features of cis-Pt(NH3)2[d(GpCpG)[ are compared with the 'bulge-out' of the ribose-trinucleotide m6(2)ApUpm6(2)A.     

The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E. coli

DNA synthesis at primers d(pT)n, d(pA)n, d(pC)n, and d(pG)n in the presence of corresponding complementary templates and at hetero-oligoprimers complementary to M13 phage DNA was investigated. The values of both -log Km and log Vmax increased linearly if homo-oligoprimers contained less than 10 nucleotides. The lengthening of d(pT)n and d(pA)n primers by one mononucleotide unit (n = 1-10) resulted in the 1.82-fold decrease of the Km values. The incremental decreases of Km for d(pC)n and d(pG)n were equal to about 2.46. The enhancement of the homo- and hetero-oligonucleotide primers' affinity to the enzyme due to one Watson-Crick hydrogen bond between complementary template and primer is about 1.35 times. This allows to calculate the Km values for primers of various structure and length up to 10 units. The objective laws of the Km and Vmax values changes for primers containing more than 10 nucleotides were analyzed.     

Specific localization of phosphointermediate filament protein in the constricted area of dividing cells

We developed antibodies pG1 and pG2 which recognize glial fibrillary acidic protein (GFAP) in its phosphorylated state. Antibodies pG1 and pG2 were produced against two synthetic peptides, Arg-Arg-Arg-Val-Thr-phosphoSer-Ala-Ala-Arg-Arg-phosphoSer (residues 3-13) and Pro-Gly-Pro-Arg-Leu-phosphoSer-Leu-Ala-Arg-Met-Pro (residues 29-39), respectively. The phosphorylation of these serine residues on the intact GFAP induces disassembly of glial filaments in vitro (Inagaki, M., Gonda, Y., Nishizawa, K., Kitamura, S., Sato, C., Ando, S., Tanabe, K., Kikuchi, K., Tsuiki, S., and Nishi, Y. (1990) J. Biol. Chem. 265, 4722-4729). Immunofluorescence and immunoblotting studies demonstrate that both antibodies react specifically with mitotic astroglial cells, thereby supporting the notion that increased phosphorylation during mitosis may directly influence intracellular organization of the glial filaments. The specific distribution pattern of the phosphoGFAP in the mitotic cells reveals that site-specific phosphorylation events may make way for the locally controlled breakdown of glial filaments in the constricted area, before the final separation of daughter cells.     

Condensation of activated diguanylates on a poly(C) template

We have studied the metal-ion catalysis of a number of reactions of the isomers of ImpGpG on a poly(C) template. In the absence of a catalytic metal ion, oligomers at least up to (pG)20 are obtained from the ImpGpG isomers in a 1-methylimidazole buffer. The Pb2+ ion improves the yield of longer oligomers and changes substantially the distribution of linkage isomers. The Pb2+ ion greatly improves the yield of longer oligomers obtained from G and ImpGpG on a poly(C) template. The self-condensation of ImpGpG in a 2, 6-lutidine buffer is much less efficient than in a 1-methylimidazole buffer. The Zn2+ greatly increases the yield of products from the [3'-5']-linked dimer, but fails to catalyze the formation of long oligomers from the [2'-5']-linked dimer. The bonds formed in the Zn2+-catalyzed self-condensation of ImpG3pG on poly(C) are mainly [3'-5']-linked.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.     

Conserved thermochemistry of guanosine nucleophile binding for structurally distinct group I ribozymes.

We report thermodynamic values for binding of the guanosine nucleophile to the ribozyme derived from the Anabaena group I intron, and find that they are similar to those measured previously for the structurally distinct Tetrahymena ribozyme. The free energy of binding guanosine 5'-monophosphate (pG) at 30 degrees C is similar for the two ribozymes. The delta(H)degrees' and delta(S)degrees' for pG binding to the Anabaena ribozyme--RNA substrate complex (E x S) are 3.4 +/- 4 kcal/mol and 27 +/- 10 e.u., respectively. The negligible enthalpic contribution and positive entropy change were found previously for the Tetrahymena ribozyme, and are considered remarkable for a hydrogen-bonding interaction between a nucleotide and a nucleic acid. These thermodynamic values may reflect conformational changes or water release upon pG binding that are comparable for the two ribozymes. In addition, the apparent chemical steps of the two ribozyme reactions share similar activation energies and a positive deltaS++. It now appears that such thermochemical values for guanosine binding and activation may be intrinsic properties of the group I intron catalytic center.     

31P NMR spectra of an oligodeoxyribonucleotide duplex lac operator-repressor headpiece complex

The interaction of a symmetric lac operator duplex, d(TGTGAGCGCTCACA)2, with the N-terminal 56-residue headpiece fragment of the lac repressor protein was monitored by 31P NMR spectroscopy. The changes in the 31P chemical shifts upon addition of the headpiece demonstrated an end point of two headpiece fragments per symmetric 14-mer duplex with each headpiece binding to the T1pG2pT3pG4pA5 ends of the duplex. The specific phosphate 31P perturbations observed are consistent with those residues implicated in protein binding by previous NMR, molecular biological, and biochemical techniques. Upon complexation, the 31P signals of phosphates G2-A5 showed upfield or downfield shifts (less than 0.2 ppm) while most other residues were unperturbed. The interactions were dependent on ionic strength. The 31P NMR data provide direct evidence for predominant recognition of the 5' strand of the 5'-TGTGA/3'-ACACT binding site.     

A two B-Z junction containing DNA resolves into an all right-handed double-helix

Natural and artificial oligonucleotides are capable of assuming many different conformations and functions. Here we present results of an NMR restrained molecular modelling study on the conformational preferences of the modified decanucleotide d((m)C1G2(m)C3G4C5(L)G6(L)(m)C7G8(m)C9G10) .d((m)C11G12(m)C13G14C15(L)G (L)16(m)C17-G18(m)C19G20 ) which contains L deoxynucleotides in its centre. This chimeric DNA was expected to form a right-left-right-handed B-type double-helix (BB*B) at low salt concentration. Actually, it matured into a fully right-handed double helix with its central C(L)pG(L) core forming a right-handed Z-DNA helix embedded in a B-DNA matrix (BZ*B). The interplay between base-base and base-sugar stackings within the core and its immediately adjacent residues was found to be critical in ensuring the stabilisation of the right-handed helix. The structure could serve as a model for the design of antisense oligonucleotides resistant to nucleases and capable of hybridising to natural DNAs and RNAs.     

Effects of dietary chemopreventive phytochemicals on P-glycoprotein function

The effects of dietary phytochemicals on P-glycoprotein function were investigated using human multidrug-resistant carcinoma KB-C2 cells and the fluorescent P-glycoprotein substrates daunorubicin and rhodamine 123. The effects of natural chemopreventive compounds, capsaicin found in chilli peppers, curcumin in turmeric, [6]-gingerol in ginger, resveratrol in grapes, sulforaphane in broccoli, 6-methylsulfinyl hexyl isothiocyanate (6-HITC) in Japanese horseradish wasabi, indole-3-carbinol (I3C) in cabbage, and diallyl sulfide and diallyl trisulfide in garlic, were examined. The accumulation of daunorubicin in KB-C2 cells increased in the presence of capsaicin, curcumin, [6]-gingerol, and resveratrol in a concentration-dependent manner. The accumulation of rhodamine 123 in KB-C2 cells was also increased, and the efflux of rhodamine 123 from KB-C2 cells was decreased by these phytochemicals. Sulforaphane, 6-HITC, I3C, and diallyl sulfide and diallyl trisulfide had no effect. These results suggest that dietary phytochemicals, such as capsaicin, curcumin, [6]-gingerol, and resveratrol, have inhibitory effects on P-glycoprotein and potencies to cause drug-food interactions.     

Double-base lesions are produced in DNA by free radicals

Evidence has been accumulating at the oligomer level that free radical-initiated DNA damage includes lesions in which two adjacent bases are both modified. Prominent examples are lesions in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized. An assay has been devised to detect double-base lesions based on the fact that the phosphoester bond 3' to a nuclesoside bearing the formamido lesion is resistant to hydrolysis by nuclease P1. The residual modified dinucleoside monophosphates obtained from a nuclease P1 (plus acid phosphatase) digest of DNA can be (32)P-postlabeled using T4 polynucleotide kinase. Using this assay the formamido single lesion and the formamido-8-oxoguanine double lesion were detected in calf thymus DNA after X-irradiation in oxygenated aqueous solution. The lesions were measured in the forms d(P(F)pG) and d(P(F)pG(H)), where P(F) stands for a pyrimidine nucleoside having the base degraded to a formamido remnant and G(H) stands for 8-oxo-deoxyguanosine. The yields in calf thymus DNA irradiated 60 Gy were 8.6 and 3.2 pmol/microgram DNA, respectively.     

Periodic acceleration: effects on vasoactive, fibrinolytic, and coagulation factors.

Cellular and isolated vessel experiments have shown that pulsatile and laminar shear stress to the endothelium produces significant release of mediators into the circulation. Periodic acceleration (pG(z)) applied to the whole body in the direction of the spinal axis adds pulses to the circulation, thereby increasing pulsatile and shear stress to the endothelium that should also cause release of mediators into the circulation. The purpose of this study was to determine whether addition of pulses to the circulation through pG(z) would be sufficient to increase shear stress in whole animals and to acutely release mediators and how such a physical maneuver might affect coagulation factors. Randomized control experiments were performed on anesthetized, supine piglets. The treatment group (pG(z)) (n = 12) received pG(z) with a motion platform that moved them repetitively head to foot at +/-0.4 g at 180 cpm for 60 min. The control group (n = 6) was secured to the platform but remained on conventional ventilation throughout the 4-h protocol. Compared with control animals and baseline, pulsatile stress produced significant increases of serum nitrite, prostacyclin, PGE(2), and tissue plasminogen activator antigen and activity, as well as D-dimer. There were no significant changes in epinephrine, norepinephrine, cortisol, and coagulation factors between groups or from baseline values. Pulsatile and laminar shear stress to the endothelium induced by pG(z) safely produces increases of vasoactive and fibrinolytic activity. pG(z) has potential to achieve mediator-related benefits from the actions of nitric oxide and prostaglandins.     

Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans

Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 10(3) to 10(4) transformants per microg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG+host9 was shown to replicate at a permissive temperature of 30 degrees C, whereas the replicon was not functional at 40 degrees C. The D. dehalogenans frdCAB operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG+host9 carrying a 0.6-kb internal frdA fragment. Single-crossover integration at the frdA locus occurred at a frequency of 3.3 x 10(-4) per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in D. dehalogenans and allow for its further exploitation as a dedicated degrader.     

Penicillin Kills Chlamydia following the Fusion of Bacteria with Lysosomes and Prevents Genital Inflammatory Lesions in C. muridarum-Infected Mice

The obligate intracellular bacterium Chlamydia exists as two distinct forms. Elementary bodies (EBs) are infectious and extra-cellular, whereas reticulate bodies (RBs) replicate within a specialized intracellular compartment termed an ‘inclusion’. Alternative persistent intra-cellular forms can be induced in culture by diverse stimuli such as IFNγ or adenosine/EHNA. They do not grow or divide but revive upon withdrawal of the stimulus and are implicated in several widespread human diseases through ill-defined in vivo mechanisms. β-lactam antibiotics have also been claimed to induce persistence in vitro. The present report shows that upon penicillin G (pG) treatment, inclusions grow as fast as those in infected control cells. After removal of pG, Chlamydia do not revert to RBs. These effects are independent of host cell type, serovar, biovar and species of Chlamydia. Time-course experiments demonstrated that only RBs were susceptible to pG. pG-treated bacteria lost their control over host cell apoptotic pathways and no longer expressed pre-16S rRNA, in contrast to persistent bacteria induced with adenosine/EHNA. Confocal and live-video microscopy showed that bacteria within the inclusion fused with lysosomal compartments in pG-treated cells. That leads to recruitment of cathepsin D as early as 3 h post pG treatment, an event preceding bacterial death by several hours. These data demonstrate that pG treatment of cultured cells infected with Chlamydia results in the degradation of the bacteria. In addition we show that pG is significantly more efficient than doxycycline at preventing genital inflammatory lesions in C. muridarum-C57Bl/6 infected mice. These in vivo results support the physiological relevance of our findings and their potential therapeutic applications.     

Daunoxome® Treatment of Solid Tumors: Preclinical and Clinical Investigations

Abstract

Daunorubicin has been entrapped into small unilamellar vesicles (50-80 nm dia) composed of a 2:1 mole ratio of highly purified DSPC:cholesterol. In earlier studies, liposomes of this size and composition had been demonstrated to deliver their entrapped contents selectively to a wide range of solid tumors in vivo. Preclinical and initial clinical investigations of these daunorubicin liposomes (DaunoXome) are discussed. In one murine solid tumor model (P1798 lymphosarcoma), a ten-fold increased delivery of entrapped daunorubicin to tumor tissue was observed. Efficacy studies in the same model indicated improved tumor regression and extended life spans that correlated with the observed degree of enhanced tumor drug delivery. In a second tumor model (MA16C mammary adenocarcinoma), a ten-fold enhancement in efficacy again was demonstrated. In terms of median survival times and long term survival rate, DaunoXome dosed at 2 mg/kg (daunorubicin) demonstrated an efficacy comparable to free drug at 20 mg/kg. Clinical pharmacokinetics paralleled findings from animal studies. In humans, DaunoXome produced daunorubicin plasma AUC levels that were more than 35-fold greater than those reported for comparable doses of free drug at 80 mg/m². Response rates above 50% have been shown for treatment of Kaposi's sarcoma. A low incidence of side effects has been observed and HIV positive patients have been able to continue antiviral therapy during DaunoXome treatments. Cardiotoxicity has not manifested clinically even for patients receiving in excess of 1 gram/m² cumulative daunorubicin.     

Template-directed extension of a guanosine 5′-phosphate covalently attached to an oligodeoxycytidylate template

We have prepared molecules in which a guanosine 5'-phosphate (pG) residue is attached to the 3' terminus of a decadeoxycytidylate (pdC)10 template via diamine linkers H2N(CH2)nNH2, n = 4-7. The pG residue acts as a primer and is extended very efficiently by incubation with activated pG derivatives to give products containing 6-9 G residues in greater than 80% yield. The detailed nature of the product distribution is discussed.     

Regulation of phospholipase D activity and ceramide production in daunorubicin-induced apoptosis in A-431 cells

We demonstrated here that daunorubicin induced apoptosis in A-431 cells, a human epidermoid carcinoma cell line. Treatment of cells with daunorubicin induced chromatin condensation, nuclear fragmentation, internucleosomal DNA degradation, and the proteolytic cleavage of PKC-delta and poly(ADP-ribose) polymerase in A-431 cells. Daunorubicin, as well as sphingomyelinase (SMase) and the exogenous cell-permeable ceramide analogue C(2)-ceramide, inhibited phospholipase D activity stimulated by phorbol 12-myristate 13-acetate or epidermal growth factor (EGF). Like ceramide, daunorubicin also decreased EGF-induced diacylglycerol generation. However, no increase in ceramide level was observed in daunorubicin-induced apoptosis in A-431 cells. Moreover, treatment of A-431 cells with exogenous cell-permeable C(2)-ceramide or SMase did not induce apoptosis. These results indicate that daunorubicin induces apoptosis in A-431 cells via a mechanism that does not involve increased ceramide formation.