Distribution of α-Tocopherol Stereoisomers in Rats

The distribution of α-tocopherol (α-Toc) stereoisomers in the tissues of rats fed on a diet containing all-rac-α-tocopheryl acetate was investigated by a newly revised HPLC. The concentrations of 2R-isomers of α-Toc in blood and tissues of the rats were significantly higher than those of 2S-isomers. In most tissues, the levels of 2S-isomers were in order SRS> (SSS +SSR)/2 > SRR.     

Is the Distribution of α-Tocopherol in Membranes Consistent with Its Putative Functions?

Vitamin E acts as an antioxidant and stabilizer of membranes. Other functions of vitamin E unrelated to its effects on membranes are emerging. Vitamin E partitions into the lipid bilayer matrix of membranes. It orients perpendicularly to the plane of the membrane with the hydroxyl group pointing to the lipid-water interface. The vitamin is not randomly distributed in the plane of the membrane but tends to form clusters. These clusters appear to be composed of vitamin E and phosphatidylcholine in a stoichiometry of about one vitamin E per 10 phospholipid molecules. Vitamin E partitions into domains of phosphatidylcholine in model membranes formed from mixtures of phosphatidylcholine and phosphatidylethanolamine irrespective of whether the phosphatidylcholine is in the fluid or gel phase. The creation of domains enriched in vitamin E in membranes is not consistent with an antioxidant function and effects on membrane structure and stability indicate other roles of the vitamin.     

Monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic Acid,a Radical Product from the Reaction of Dehydro-l-ascorbic Acid with an α-Amino Acid

One of the radical species produced by the reaction of dehydro-l-ascorbic acid with an α-amino acid gave a very characteristic hyperfine structure in its electron spin resonance spectrum. The same spectrum was also obtained when l-scorbamic acid was oxidized with some oxidants, indicating the formation of the radical via the oxidation of l-scorbamic acid. From the results of deuterium exchange experiments, simulation spectra and the reduction of 2,2′-nitrilodi-2(2′)-deoxy-l-ascorbic acid monoammonium salt, the radical was concluded to be monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic acid. Possible formation mechanism of the radical was also discussed.     

Complexes of platinum(II) containing 2,2′-bithiazoline and 2,2′-bipyrimidine as binucleating ligands

Dimethyl and diphenyl platinum(II) complexes containing binucleating α-diimine ligands BN (BN = 2,2′-bithiazoline and 2,2′-bipyrimidine) have been isolated and characterized. Electrophilic attack of mercuric chloride on the mononuclear compounds leads to binuclear systems of C_2v symmetry, with the two chelating moieties of the ligands occupied by platinum and mercury, respectively. ¹H NMR spectroscopy suggests a large transmission of electronic effects between the metals through the ligands.     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

Complexes of magnesium(II) and other divalent metal ions with adenosine 5′-triphosphate and 2,2′-dipyridylamine in aqueous solution

Binary and ternary systems involving adenosine 5′-triphosphate (ATP), 2,2′-dipyridylamine (DPA) and magnesium, calcium, strontium, manganese, cobalt, copper, and zinc(II) metal ions have been investigated in aqueous media by potentiometric titrations. The analysis of the titration curves shows the existence of M(ATP)²⁻, M(ATP)(H)⁻, and M(ATP)₂(H)₂⁴⁻ species for alkaline-earth metal ions, while no ternary complex can be detected. For transition metal ions both binary and ternary species are found. Binary M(ATP)₂(H)₂⁴⁻ complexes are present in solutions containing manganese and cobalt(II) metal ions but these species cannot be revealed in the case of copper and zinc(II). Ternary complexes as M(ATP)(DPA)²⁻ and M(ATP)(DPA)(H)⁻ are common to all transition metals. Binuclear and hydroxo complexes as M₂(ATP)(OH)⁻ and M(ATP)(OH)³⁻ are found only for copper and zinc(II). A hypothesis on the possible role of the species M-ATP in 1:2 ratio in the dephosphorylation mechanism is advanced on the basis of a comparison between the equilibrium data in the solution phase and the solid state structures of the magnesium, calcium, and manganese(II)- ATP-DPA systems.     

Colorimetric Determination of α-Amino Nitrogen in Urine and Plasma with Ninhydrin Reaction

Bacillus stearothermophilus TH 6–2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a functional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosine, the primary substrate of Pu-NPase II. The Pu-NPase I gene of TH 6–2 has been cloned, sequenced, and expressed in E. coli. The gene corresponded to an open reading frame of 822 nucleotides that translates into a putative 274-amino acid protein with a molecular weight of 29,637. The deduced amino terminus sequence completely coincided with that found for the purified enzyme. The cloned gene was overexpressed in E. coli by using the trc promoter to produce an active enzyme in large quantities. The amino acid sequence of Pu-NPase I shared 50% similarity with those of human and mouse purine nucleoside phosphorylases.     

Light-Induced Lipid Peroxidation in Isolated Chloroplasts and Role of α-Tocopherol

Lipid peroxidation in isolated chloroplasts illuminated by visible light and the role of α-tocopherol in chloroplasts were studied. The TBA reactants and fluorescent products derived from lipid peroxidation were formed by illumination. Peroxidation was inhibited by free radical scavengers and ¹O₂ quenchers. Hydroxy methyl octadecanoates, which were the reduced and hydrogenated products of lipid hydroperoxides, were detected. Among them, 10-and 15-hydroxy methyl octadecanoates were generated from ¹O₂ oxidation. On the other hand, lipid hydroperoxides did not accumulate in this peroxidation process. The amount of α-tocopherol in the chloroplasts decreased with lipid peroxidation, and α-tocopheryl quinone was produced. The results indicate that α-tocopherol acts as a free radical scavenger for photo-oxidation of chloroplasts.     

Reaction of 3′,5′-di-O-acetyl-2′-deoxyguansoine with hypobromous acid

Hypobromous acid (HOBr) is formed by eosinophil peroxidase and myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^? in the host defense system of humans, protecting against invading bacteria. However, the formed HOBr may cause damage to DNA and its components in the host. When a guanine nucleoside (3′,5′-di-O-acetyl-2′-deoxyguansoine) was treated with HOBr at pH 7.4, spiroiminodihydantoin, guanidinohydantoin/iminoallantoin, dehydro-iminoallantoin, diimino-imidazole, amino-imidazolone, and diamino-oxazolone nucleosides were generated in addition to an 8-bromoguanine nucleoside. The major products were spiroiminodihydantoin under neutral conditions and guanidinohydantoin/iminoallantoin under mildly acidic conditions. All the products were formed in the reaction with HOCl in the presence of Br^?. These products were also produced by eosinophil peroxidase or myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^?. The results suggest that the products other than 8-bromoguanine may also have importance for mutagenesis by the reaction of HOBr with guanine residues in nucleotides and DNA.     

2,2′- bipyridine coplanar with coordination square of Pd(II) nonyldithiocarbamato antitumor complex interacting with DNA in two distinct steps

Cisplatin is one of the most effective chemotherapy drugs, and has been widely employed for more than four decades in the treatment of different forms of human tumors. In recent years, various examples of metal complex-based compounds have been used for medicinal purposes. In this context, the novel palladium(II) complex, [Pd(non-dtc)(bpy)]NO₃, (non-dtc = nonyldithiocarbamate and bpy = 2,2′- bipyridine) has been synthesized and characterized by means of elemental analysis, conductivity measurements, FT-IR, ¹H NMR, ¹³C NMR, and electronic spectroscopy studies. The 50% cytotoxic concentrations (Ic₅₀) of this Pd(II) complex (0.53 mM) and cisplatin (154 mM) against human cell tumor line (K562) indicates its interaction with DNA of cancer cell at quite low concentration. Thus, binding characteristics of this compound to calf thymus DNA (CT-DNA) has been investigated by UV–vis absorption spectroscopy and fluorescence spectra. The exciting observation of this work in the UV–visible studies was that the Pd(II) complex exhibit two or more types of interaction with CT-DNA. Such properties have rarely been observed in the literature. This complex cooperatively binds with DNA and denatures it too. Fluorescence studies proved the intercalation mode of binding and the other modes seems to be hydrophobic and electrostatic interactions. Binding parameters and thermodynamics of the interaction with CT-DNA are also described. Finally, multifunctional interactions of [Pd(non-dtc)(bpy)]NO₃ make it suitable to interact with DNA of cancer cell at quite low concentration and if it is used as anticancer agent, very low doses will be needed which may have fewer side effects.     

Reaction of Toxic Bicyclic Phosphates with Acetylcholinesterases and α-Chymotrypsin

Some toxic bicyclic phosphates (BPs) inhibited acetylcholinesterases (AChEs), but the activity was very weak. Even the most potent inhibitor, 4-nitro BP, inhibited bovine erythrocyte and housefly head AChEs by only 37 and 38 per cent, respectively, at 1.5 mm. Kinetic analysis indicated that the poor inhibitory activity of 4-nitro BP is ascribed not only to the low affinity for AChEs but also to its poor phosphorylating ability. Similar findings were obtained in the case of the reaction of BPs with the serine enzyme α-chymotrypsin. Despite the relatively high reactivity in an alkaline solution, BPs are much less active than other bioactive organophosphorus esters in phosphorylating a general-base-catalyzed hydroxyl group. This fact suggests that the toxic action of BPs does not result from the phosphorylation of a critical site in biological systems.     

Crystal Structure of an EAL Domain in Complex with Reaction Product 5′-pGpG

FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5′-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5′-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.     

Evaluation of Serum and Tissue Levels of α-Tocopherol

This study was designed to test the effect of supplementation of several antioxidants, including α-tocopherol, on the clinical reduction of premalignant oral lesions. Samples of oral mucosa and serum were taken from baseline to 9 months of supplementation from patients with premalignant oral lesions and analyzed for α-tocopherol by HPLC. Statistical increases in both serum and tissue α-tocopherol were found after supplementation. There was no statistical relationship between α-tocopherol and β-carotene levels.     

Syntheses of α-Tocopherol and Vitamin K1

α-Tocopherol and vitamin K₁ were synthesized by using 3,7,11,15-tetramethylhexa- decane-1,3-diol instead of phytols.     

Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.     

Efficient Synthesis of 2′-Bromo-2′-Deoxy-3′,5′-O-TPDS-pyrimidine Nucleosides by Boron Trifluoride Catalyzed Reaction of O2,2′-Anhydro-(1-β-D-Arabinofuran0Syl)pyrimidine Nucleosides with Lithium Bromide

Abstract

Efficient syntheses of 2′-bromo-2′-deoxy-3′,5′-O-TPDS-uridine (5a) and 1-(2-bromo-3,5-O-TPDS-β-D-ribofuranosyl)thymine (5b) from uridine and 1-(β-D-ribofuranosyl)thymine are described, respectively. The key step is a treatment of 3′,5′-O-TPDS-O²,2′-anhydro-1-(β-D-ardbinofuranosyl)uracil (4a) and -thymine (4b) with LiBr in the presence of BF₃-OEt₂ in 1,4-dioxane at 60°C to give 5a and 5b in 98%, and 96% yield, respectively.

     

Effects of α-Tocopherol on Carbon Tetrachloride Metabolism in Rat Liver Microsomes

α-tocopherol is the major lipid-soluble radical-scavenging antioxidant in rat liver. It has long been used as a putative protective agent in CC1₄ induced liver injury but with variable results. We have used a-tocopherol loaded rat liver microsomes to study the effect of this vitamin on CC1₄ metabolism in vitro. As expected, a-tocopherol inhibits CC1₄-dependent microsomal lipid peroxidation and, at a very high concentration, will inhibit the covalent binding of CC1,- to microsomal protein by up to 50%. No inhibitory effect was observed towards CC1₃ production as measured by the electron spin resonance technique of spin-trapping but this apparent discrepancy may represent a limitation of the technique. The high levels required to inhibit covalent binding probably preclude the likelihood of a-tocopherol significantly affecting that phenomenon at endogenous concentrations but may be relevant to other experiments employing high doses of a-tocopherol as an experimental hepatoprotective agent.