In vitro competition between adenosine(5')tetraphospho(5')adenosine and deoxyribonucleic acid in the reaction with diamminedichloroplatinum(II)

Competition between adenosine(5')tetraphospho(5')adenosine (Ap4A) and DNA for the synthesis of adducts with the cis or trans isomer of diamminedichloroplatinum(II) was measured in the presence and absence of magnesium and spermidinium ions. Reaction products were analysed by circular dichroism, poly(ethyleneimine) thin-layer chromatography and reversed-phase chromatography. Competition was affected by the oligovalent cations that bound specifically to the dinucleotide. Platination of DNA was favoured under all conditions. Chromatin was less competitive. The mechanism was kinetic competition, DNA reacting considerably faster than Ap4A. Platinum(II) did not exchange between adducts and free DNA and Ap4A, respectively. On that basis only low amounts of Ap4A adducts were estimated to be formed under conditions of clinical chemotherapy. The cis and trans isomers of diamminedichloroplatinum(II) were equally effective. Platinum(II) adducts of Ap4A were neither degraded by Ap4A-specific pyrophosphohydrolases nor by phosphodiesterase nor in the presence of unfractionated extract of calf thymus. Unphysiologically high concentrations of Crotalus durissus phosphodiesterase I were required for hydrolytic splitting, the amount of which was similar for both platinum(II) isomer adducts. The results suggest that Ap4A platinum(II) adducts might accumulate during chemotherapy of cancer treatment.     

2' (or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-triphosphate as a probe for the binding site of heavy meromyosin ATPase.

1. From NMR, IR and visible absorption studies of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine (TNP-Ad(, and 1-(2'-hydroxyethoxy)-2, 4, 6-trinitrobenzene (TNP-EG), it was concluded that there is an intramolecular interaction between the base and 2, 4, 6-trinitrophenyl (TNP) moieties in the TNP-ATP molecule. 2. A broad new absorption band was observed in the 530-630 nm region when excess indole was added to reaction mixtures containing TNP-ATP dissolved in 50% methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, TNP-ATP and TNP-Ad underwent spectral shifts in the 400-550 nm region. The formation of a 1:1 complex apparently occurred between TNP-ATP and aromatic amino acid derivatives, and the complex with N-acetyltryptophan was stable in 50% methanol. The difference spectrum of TNP-EG vs. TNP-ATP closely resembled that induced by the addition of N-acetyltryptophan to the TNP-ATP solution. 3. The binding of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) to heavy meromyosin (HMM) was studied by the rapid gel equilibrium method using Sephadex G-25. A dissociation constant of 1.4 muM and a maximum binding number of 1.8 were obtained in 0.15 M KCl, 10 mM MgCl2, and 50 mM Tris-HCl (pH 8.0) at 25 degrees. TNP-ADP bound to the enzyme caused a characteristic spectral shift in the visible region. This spectral shift was explained in terms of an interaction between tryptophanyl residues and the adenine base of TNP-ADP bound to the enzyme. TNP-ADP quenched the tryptophanyl fluorescence, but TNP-EG and TNP-Ad did not. In the presence of 6 M guanidine hydrochloride, TNP-ADP scarcely quenched the tryptophanyl fluorescence, its effect being comparable to that of TNP-Ad.     

Phosphofructokinase (isozymes) activation by theophylline or by 3',5' cyclic AMP administration.

Phosphofructokinase activity of rat thyroid, kidney and muscle can be enhanced by in vivo administration of theophylline or cyclic AMP. This enhancement occurs in the different tissues to a different extent depending on the tissue-specific isozymes. Thyroid and muscle phosphofructokinase which is mainly A-type, is strongly stimulated after administering theophylline in the drinking water over a 8 days period. The kidney enzyme which encompasses about 50% of each A and B type is activated to a lesser extent. Liver phosphofructokinase which is pure B-type shows very little response if any. Comparable results have been obtained with either the muscle or liver enzyme after two to four hours treatment with a single injection of 10 mg/rat of either cyclic AMP or theophylline. This short-term activation could be eliminated by treating the animals with Actinomycin D, 30 min prior to theophylline or cyclic AMP. The possible mechanisms leading to the enhancement of phosphofructokinase activity are discussed.     

Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.     

Adenosine(5')hexaphospho(5')adenosine stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum.

Stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum by various adenosine(5')oligophospho(5')adenosines (ApnA, n = 2-6) by a rapid quenching technique using radioactive calcium was studied. Ap4A, Ap5A and Ap6A, as well as adenosine 5'-[beta, gamma-methylene]triphosphate (AdoPP [CH2]P), a non-hydrolyzable ATP analogue, stimulated the Ca(2+)-release channel, whereas Ap2A and Ap3A had no effect. At a concentration of 0.5 mM, the order of stimulation was AdoPP[CH2]P less than Ap4A less than Ap5A much less than Ap6A. As well as having the highest affinity (0.44 mM for half-maximal stimulation), Ap6A showed an extraordinarily high Hill coefficient of 3.3 (1.9 for AdoPP[CH2]P, 2.1 for Ap5A). The stimulating effect of Ap6A was reversible, yet its dissociation proceeded very slowly. Stimulation of Ca2+ release by Ap6A was counteracted by Mg2+ and ruthenium red. A 2',3'-dialdehyde derivative of Ap6A, which is a chemical probe for amino groups, stimulated irreversibly the Ca(2+)-release channel and modified some high-molecular-mass sarcoplasmic reticulum proteins, possibly including the channel protein. Our data suggest that Ap6A stimulates the Ca2+ channel by binding to the activation site of the channel subunit and simultaneously preventing the spontaneous decay of the Ca2+ channel by keeping together two of the four channel subunits by bridging them with its two adenosine groups.     

5-Bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine and -thymidine derivatives with potent antiviral activity

Synthesis, antiviral, and cytotoxic activities of 5-bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine (4,5) and -thymidine (6,7) are reported. Compounds 4 and 5 exhibited a broad spectrum of antiherpes activity against (HSV-1, HSV-2, HCMV, and VZV).     

Induction of 2',5' oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus or poly(I)poly(C)

Infection of 13 month-old C3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2',5' oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.     

Stoichiometric binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate to bovine heart cytochrome c oxidase.

The binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP-ATP per monomer COX with a Kd of 1.6 microM.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

Synthesis of 5'(3')-O-amino nucleosides

Synthetic methods leading to 5'(3')-O-amino nucleosides have been developed in an effort to prepare derivatives that may have antitumor or antiviral activities. They are based on ring opening of O2,5'-cyclonucleosides with the N-protected hydroxylamines and dehydrative coupling of 5'(3')-O-unprotected nucleosides with N-hydroxyphthalimide.     

Characterization of the aac(6')-Ik gene of Acinetobacter sp. 6

Abstract Chromosomal DNA of different species of mycobacteria, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium and Mycobacterium smegmatis , has been submitted to polymerase chain reaction using two oligonucleotide primers highly homologous to DNA sequences flanking the quinolone resistance-determining region in the gyrA gene of Escherichia coli and Staphylococcus aureus . For each of these mycobacterial species, a 150-bp DNA fragment hybridizing with an intragenic probe of the gyrA gene of E. coli K12 was obtained. The nucleotide sequences of the 108-bp fragments amplified from M. tuberculosis and M. avium were determined. The two sequences were 87% homologous. Except for one residue, their deduced amino acid sequences were identical and shared 67% homology with the quinolone resistance-determining region of the gyrase A subunits of E. coli and S. aureus . Sequencing of the 108-bp fragment amplified from an in vitro mutant of M. avium , highly resistant to fluoroquinolones, showed a point mutation leading to the substitution of Ala for Val at a position corresponding to residues involved in quinolone resistance in E. coli and S. aureus , i.e. Ser 83 for E. coli and Ser 84 for S. aureus .     

The kinetics and specificity of the reaction of 2'(3')-O-bromoacetyluridine with bovine pancreatic ribonuclease A.

2'(3')-O-Bromoacetyluridine reacts rapidly and selectively with bovine pancreatic ribonuclease A at pH 5.5 and 25 degrees. Under conditions of high molar ratios of nucleoside derivative to enzyme, the only derivative is N-3-carboxymethylhistidine-12 ribonuclease A. The reaction occurs almost exclusively with the histidine-12 residue at the active site inactivation of the enzyme is accompanied by the stoichiometric disappearance of unmodified ribonuclease A and appearance of the product, N-3-carboxymethylhistidine-12 ribonuclease A. Kinetic studies indicate a mechanism involving saturation of the enzyme by the nucleoside derivative. The inhibitor constant, Kb, is 0.087 M and k3 is 35.1 times 10(-4) sec minus 1. The reaction of 2'(3')-O-bromoacetyluridine with the enzyme occurs at a rate approximately 3100 times greater than that corresponding to the reaction with L-histidine. The alkylation reaction is inhibited competitively by uridine with a Ki of 0.013 M. 2'(3')-O-Bromoacetyluridine inactivates ribonuclease A 4.5 times faster than bromoacetic acid and the specificity for alkylation of active-site histidine residues is different. 2'(3')-O-Bromoacetyluridine reacts 1000 times more rapidly with ribonuclease A than iodoacetamide. The contribution of nucleoside binding to the overall rate of alkylation is discussed.     

Rapid amplification of 5' complementary DNA ends (5' RACE)

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.     

Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells.

Four estrogen receptor-positive (ER+) [MCF-7, T47D, ZR75 and BT474] and 3 ER- [Hs578T, MDA-MB-468 and MDA-MB-231] human breast cancer cell lines were examined for expression of the IGFBP-5 and IGFBP-6 genes. Northern blot analysis revealed that all cell lines, except MDA-MB-231, expressed IGFBP-5 mRNA. IGFBP-6 mRNA, however, was expressed only by the ER- cell lines. Western immunoblotting indicated that the previously unidentified 31-kDa and 32-kDa IGF binding species secreted by these cell lines are IGFBP-5. The levels of IGFBP-4 and IGFBP-5 were increased in MCF-7 cells by estradiol and IGF-I, respectively, indicating that these BPs may contribute to the growth stimulatory response to these mitogens.     

Steric and charge factors in the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase hydrolysis.

The contribution of steric and negative charge factors to the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase was investigated. The hydrolysis rates of uridylyl(3'-5')N6-(N-threonylcarbonyl)-adenosine, its model derivatives, methyl ester and O-benzyl ester, together with unmodified uridyly (3'-5')adenosine, were studied. It was found that the contribution of both factors is of the same order. The steric inhibition of digestion is distinctly higher than that confirmed by N6-(delta2-isopentenyl)adenosine [1], which is ascribed to the rigid conformation of the threonylcarbonyladenosine side chain.