DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the (32)P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a (32)P-postlabeling/thin layer chromatography (TLC) method and a (32)P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36-36.4 microM) for 3h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N(6)-C2-ABA) and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N(2)-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3'p-N(6)-C2-ABA and its relative adduct labeling (RAL) value at 36.4 microM of 3-NBA was 200.8+/-86.1/10(8)nucleotide.     

Postlabelling analysis of DNA adducts formed in human hepatoma cells treated with 3-nitrobenzanthrone

3-Nitrobenzanthrone (NBA) is one of the most mutagenic nitroaromatic compounds that has been found recently in diesel exhaust and airborne particles. A [³²P]-postlabelling analysis was carried out to examine the adducts in DNA from human hepatoma HepG2 cells treated with NBA. Two major and two minor adduct spots were obtained in the analysis. The structure of the compound obtained from one of the minor adduct spots was identified to be N-acetyl-3-amino-2-(2′-deoxyguanosin-3′,5′-bisphosphate-8-yl)-benzanthrone, based on identical mobility of the compound with that of synthetic standards in thin-layer chromatography and high performance liquid chromatography. This substance is the identical adduct found in our previous in vitro study. The yet-unidentified major adduct spots may be guanosin- and adenosin-benzanthrone adducts without the N-acetyl group.     

Improved high-performance liquid chromatography analysis of P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification

An improved HPLC-based ³²P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C₁₈ precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10⁷ bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10⁴ to ≈ 2±1 adducts per 10⁹ bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3′-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the PhIP-DNA adducts, this method can be adjusted for analysis of other DNA adducts and is readily automated for high throughput.     

DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation.     

DNA sequence dependence of guanine-O alkylation by the N-nitroso carcinogens N-methyl- and N-ethyl-N-nitrosourea

After intracellular in vitro exposure to the mutagenic and carcinogenic N-nitroso compounds N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), respectively, the average relative amounts of the premutational lesion O⁶-alkylguanine represent about 6% and 8% of all alkylation products formed in genomic DNA. At the level of individual DNA molecules gunine-O⁶ alkylation does nor occur at random; rather, the probability of a substitution reaction at the nucleophilic O⁶ atom is influenced by nucleotide sequence, DNA conformation, and chromatin structure. In the present study, 5 different double-stranded polydeoxynucleotides and 15 double-stranded oligodeoxynucleotides (24-mers) were reacted with MeNU or EtNU in vitro under standardized conditions. Using a competitive radioimmunoassay in conjunction with an anti-(O⁶-2′-deoxyguanosine) monoclonal antibody, the frequency of guanine-O⁶ alkylation was found to be strongly dependent on the nature of the nucleotides flanking guanine on the 5t́ and 3′ sides. Thus, a 5′ neighboring guanine, followed by 5t́ adenine and 5′ cytosine, provided an up to 10-fold more ‘permissive’ condition for O⁶-alkylation of the central guanine than a 5′ thymine (with a 5-methylcytocine in the 5′ position being only slightly less inhibitory). Thymine and cytosine were more ‘permissive’ when placed 3′ in comparison with their affects in the 5′ flanking position.     

Solution structure investigations of olefin Pd(II) and Pt(II) complex ion pairs bearing α-diimine ligands by F, H-HOESY NMR

Complexes [M(η¹,η²-C₈H₁₂OMe)((2,6-(R)₂---C₆H₃)N=C(R′)---C(R′)=N((2,6-(R)₂---C₆H₃))]PF₆ (where M=Pd, R=H and R′₂=Me₂ (1), M=Pd, R=Me and R′₂=Me₂ (2), M=Pd, R=Et and R′₂=Me₂ (3), M=Pd, R=iPr and R′₂=Me₂ (4), M=Pd, R=iPr and R′₂=An (5), M=Pt, R=iPr and R′₂=An (6)) were synthesized by the reaction of [M(η¹,η²-C₈H₁₂OMe)Cl]₂ with the appropriate α-diimine ligand in the presence of NH₄PF₆. Their ion pair structure in solution was investigated by detecting dipolar interactions between protons belonging to the cation and fluorine nuclei of the anion (interionic contacts) in the ¹⁹F, ¹H-HOESY NMR spectra. In complexes 1–4, the anion in solution is located close to the peripheral protons of the α-diimine ligand and it interacts with the R′ protons and with the R protons that point toward the R′ groups. The steric protection of apical position exerted by the R substituents is clearly illustrated by the absence of interionic contacts between any protons of the cycloctenylmethoxy-moiety and the anion for R≥Me in 1–4. In complexes 5 and 6 the interactions between the anion and the peripheral N,N protons also predominate but other anion–cation orientations are significantly present and, consequently, the interionic structure is less specific.     

Calcium regulation of guanine nucleotide activation of hepatic adenylate cyclase

Pretreatment of isolated rat liver plasma membranes by washing with NaHCO₃ buffer or by exposure to the chelator ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5′-(β-γ-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca²⁺, Mg²⁺ or Mn²⁺. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca²⁺. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca²⁺ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P.Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca²⁺-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca²⁺. The effects of low concentrations (less than 100 μM) of the phenothiazine could be reversed totally by Ca²⁺ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca⁺ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.     

Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl⁻¹) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl⁻¹ molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l⁻¹ concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l⁻¹ concentration.

The scientific relevance of the paper

The azo dye Methyl red (100 mg l⁻¹) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants.     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Aromatic amines and nitroarenes are important antioxidants and intermediates in the synthesis of dyes, pesticides and plastics. In the present paper we introduce methods for the synthesis of deuterated standards: 3-[²H₈]aminofluoranthene, 3,3′-dimethyl-[²H₄]benzidine, [²H₄]benzidine, N′-acetyl-[²H₄]benzidine, 2,4-[²H₆]toluenediamine, 2,6-[²H₆]toluenediamine. These standards have been used for the quantification of haemoglobin adducts of diamines and polyaromatic amines. Haemoglobin was hydrolysed in 0.1 M sodium hydroxide and the hydrolysate extracted with dichloromethane. The extracts were derivatised with heptafluorobutyric anhydride and analysed by GC–MS with negative chemical ionisation. In one run up to 15 aromatic amines can be determined: 6-aminochrysene, 3-aminofluoranthene, 2-aminofluorene, 1-aminopyrene, benzidine, 3,3′-dichlorobenzidine, 3,3′-dimethoxybenzidine, 3,3′-dimethylbenzidine, 3,3′-methylenedianiline, 4,4′-methylenedianiline, N′-acetyl-benzidine, N′-acetyl-4,4′-methylenedianiline, 4,4′-methylene bis(2-chloroaniline), 2,4-toluenediamine and 2,6-toluenediamine.     

Haemoglobin adducts of aromatic amines: diamines and polyaromatic amines

Aromatic amines and nitroarenes are important antioxidants and intermediates in the synthesis of dyes, pesticides and plastics. In the present paper we introduce methods for the synthesis of deuterated standards: 3-[²H₈]aminofluoranthene, 3,3′-dimethyl-[²H₄]benzidine, [²H₄]benzidine, N′-acetyl-[²H₄]benzidine, 2,4-[²H₆]toluenediamine, 2,6-[²H₆]toluenediamine. These standards have been used for the quantification of haemoglobin adducts of diamines and polyaromatic amines. Haemoglobin was hydrolysed in 0.1 M sodium hydroxide and the hydrolysate extracted with dichloromethane. The extracts were derivatised with heptafluorobutyric anhydride and analysed by GC–MS with negative chemical ionisation. In one run up to 15 aromatic amines can be determined: 6-aminochrysene, 3-aminofluoranthene, 2-aminofluorene, 1-aminopyrene, benzidine, 3,3′-dichlorobenzidine, 3,3′-dimethoxybenzidine, 3,3′-dimethylbenzidine, 3,3′-methylenedianiline, 4,4′-methylenedianiline, N′-acetyl-benzidine, N′-acetyl-4,4′-methylenedianiline, 4,4′-methylene bis(2-chloroaniline), 2,4-toluenediamine and 2,6-toluenediamine.     

Methylenedioxy- and methoxyflavones from Melicope coodeana syn. Euodia simplex

Three new natural products, 3,8-dimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone, 3,6,8-trimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone and 3,6,8,3′,4′-pentamethoxy-5,7-dihydroxyflavone were isolated from Melicope coodeana syn. Euodia simplex (Rutaceae) along with 3,6,3′-trimethoxy-5,7,4′-trihydroxyflavone and 3,3′-dimethoxy-5,7,4′-trihydroxyflavone. The structural assignments are based on ¹H and ¹³C NMR data, including discussion of the chemical shifts of C-2 in 3,5-dihydroxy- and 3-methoxy-5-hydroxyflavones. The presence of highly methoxylated and methylenedioxyflavones is characteristic of the genus Melicope, and the present findings support the recent transfer of Euodia simplex to Melicope.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Ecophysiology of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae) in Moreton Bay, Australia

Large blooms of the marine cyanobacterium Lyngbya majuscula in Moreton Bay, Australia (27°05′S, 153°08′E) have been re-occurring for several years. A bloom was studied in Deception Bay (Northern Moreton Bay) in detail over the period January–March 2000. In situ data loggers and field sampling characterised various environmental parameters before and during the L. majuscula bloom. Various ecophysiological experiments were conducted on L. majuscula collected in the field and transported to the laboratory, including short-term (2 h) ¹⁴C incorporation rates and long-term (7 days) pulse amplitude modulated (PAM) fluorometry assessments of photosynthetic capacity. The effects of L. majuscula on various seagrasses in the bloom region were also assessed with repeated biomass sampling. The bloom commenced in January 2000 following usual December rainfall events, water temperatures in excess of 24 °C and high light conditions. This bloom expanded rapidly from 0 to a maximum extent of 8 km² over 55 days with an average biomass of 210 g_dw⁻¹ m⁻² in late February, followed by a rapid decline in early April. Seagrass biomass, especially Syringodium isoetifolium, was found to decline in areas of dense L. majuscula accumulation. Dissolved and total nutrient concentrations did not differ significantly (P > 0.05) preceding or during the bloom. However, water samples from creeks discharging into the study region indicated elevated concentrations of total iron (2.7–80.6 μM) and dissolved organic carbon (2.5–24.7 mg L⁻¹), associated with low pH values (3.8–6.7). ¹⁴C incorporation rates by L. majuscula were significantly (P < 0.05) elevated by additions of iron (5 μM Fe), an organic chelator, ethylenediaminetetra-acetic acid (5 μM EDTA) and phosphorus (5 μM PO₄⁻³). Photosynthetic capacity measured with PAM fluorometry was also stimulated by various nutrient additions, but not significantly (P > 0.05). These results suggest that the L. majuscula bloom may have been stimulated by bioavailable iron, perhaps complexed by dissolved organic carbon. The rapid bloom expansion observed may then have been sustained by additional inputs of nutrients (N and P) and iron through sediment efflux, stimulated by redox changes due to decomposing L. majuscula mats.     

Protective effects of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon: Removal of O-methylguanine DNA adducts, p53 expression, inducible nitric oxide synthase downregulation and apoptotic induction

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 μmol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O⁶-methylguanine (O⁶-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P < 0.05), and colonic (at three sequential time points; ANOVA, F = 4.96, P < 0.05) O⁶-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P < 0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P = 0.0026, r = 0.83, r² = 0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.     

No carrier added synthesis of O-(2'-[18F]fluoroethyl)-L-tyrosine via a novel type of chiral enantiomerically pure precursor, NiII complex of a (S)-tyrosine Schiff base

O-(2′-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) has gained much attention as a promising amino acid radiotracer for tumor imaging with positron emission tomography (PET) due to favorable imaging characteristics and relatively long half-life of ¹⁸F (110 min) allowing remote-site application. Here we present a novel type of chiral enantiomerically pure labeling precursor for [¹⁸F]FET, based on NiII complex of a Schiff’s base of (S)-[N-2-(N′-benzylprolyl)amino]benzophenone (BPB) with alkylated (S)-tyrosine, Ni-(S)-BPB-(S)-Tyr-OCH2CH2X (X = OTs (3a), OMs (3b) and OTf (3c)). A series of compounds 3a–c was synthesized in three steps from commercially available reagents. Non-radioactive FET as a reference was prepared from 3a in a form of (S)-isomer and (R,S) racemic mixture. Radiosynthesis comprised two steps: (1) n.c.a. nucleophilic fluorination of 3a–c (4.5–5.0 mg) in the presence of either Kryptofix 2.2.2.or tetrabutylammonium carbonate (TBAC) in MeCN at 80 °C for 5 min, followed by (2) removal of protective groups by treating with 0.5 M HCl (120 °C, 5 min). The major advantages of this procedure are retention of enantiomeric purity during the ¹⁸F-introduction step and easy simultaneous deprotection of amino and carboxy moieties in 3a–c. Radiochemically pure [¹⁸F]FET was isolated by semi-preparative HPLC (C18 μ-Bondapak, Waters) eluent aq 0.01 M CH₃COONH₄, pH 4/C₂H₅OH 90/10 (v/v). Overall synthesis time operated by Anatech RB 86 laboratory robot was 55 min. In a series of compounds 3a–c, tosyl derivative 3a provided highest radiochemical yield (40–45%, corrected for radioactive decay). Enantiomeric purity was 94–95% and 96–97%, correspondingly, for Kryptofix and TBAC assisted fluorinations. The suggested procedure involved minimal number of synthesis steps and suits perfectly for automation in the modern synthesis modules for PET radiopharmaceuticals. Preliminary biodistribution study in experimental model of turpentine-induced aseptic abscess and Glioma35 rat’s tumor (homografts) in Wistar rats has demonstrated the enhanced uptake of radiotracer in the tumor area with minimal accumulation in the inflamed tissues.     

Relation between red cell membrane (Na + K)-ATPase and band 3 protein

This study is designed to examine the participation of the major red cell membrane protein, band 3 protein, in the chain which transmits information from the cardiac glycoside site on the external face of the cell (Na⁺ + K⁺)-ATPase to the megadalton glycolytic enzyme complex within the cell. The experiments show that the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, affects the resonance of 2,3-diphosphoglycerate, as does the cardiac glycoside cation transport inhibitor, ouabain. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor which indicates that there is coupling in the red cell between the (Na⁺ + K⁺)-ATPase and band 3 protein. Band 3 protein was separated from the membrane and partially purified following the technique of Yu and Steck ((1975) J. Biol. Chem. 250, 9170–9175). When glyceraldehyde-3-phosphate dehydrogenase was added to the separated band 3 protein preparation, addition of cardiac glycosides caused shifts in the ³¹P resonance of glyceraldehyde 3-phosphate. These experiments indicate that there is coupling between the (Na⁺ + K⁺)-ATPase and band 3 protein in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell.     

Methacholine-induced cGMP production is regulated by nitric oxide generation in rabbit submandibular gland cells

Guanosine 3′,5′-monophosphate (cGMP) is an intracellular messenger in various kinds of cell. We investigated the regulation of cGMP production by nitric oxide (NO) in rabbit submandibular gland cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose- and time-dependent manner, but the α-agonist phenylephrine, substance P and the β-agonist isoproterenol failed to evoke cGMP production. In fura-2-loaded cells, methacholine induced an increase in intracellular Ca²⁺ ([Ca²⁺]_i) in a concentration-dependent manner, which was similar to that for cGMP production. When the external Ca²⁺ was chelated with EGTA, methacholine failed to induce cGMP production. Ca²⁺ ionophore A23187 and thapsigargin, which induce the increase in [Ca²⁺]_i without activation of Ca²⁺-mobilizing receptors, mimicked the effect of methacholine. cGMP production induced by methacholine, A23187 and thapsigargin was clearly inhibited by N^G-nitro- -arginine methylester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS). S-Nitroso-N-acetyl- -penicillamine (SNAP), a NO donor, induced cGMP formation. In the lysate of rabbit submandibular gland cells, Ca²⁺-regulated nitric oxide synthase activity was detected. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is regulated by NO generation via the increase in [Ca²⁺]_i.     

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ⁸′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.     

Glial tumor cells deficient in thymidine kinase: isolation and characterization

Rat astrocytoma cells were treated with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine and grown in the presence of high concentrations of the thymidine analog 5-bromo-2′-deoxyuridine. A surviving clone, designated C₆TK⁻, lacked thymidine kinase but exhibited differentiated functions such as catecholamine-sensitive adenylate cyclase activity, cortisol-inducible glycerol-1-phosphate dehydrogenase activity, and S-100 protein biosynthesis.