Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

In vitro metabolism of N-methyl-dibenzo [c,g]carbazole a potent sarcomatogen devoid of hepatotoxic and hepatocarcinogenic properties

The metabolism of N-methyl substituted 7H-dibenzo[c,g]carbazole (N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH-N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25-30%) and 3-OH-N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC.     

Effects of newly synthesised platinum(ii) complexes on mitochondrial functions

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum(II)diamminodichloride (cis-PDD). Survival after treatment determined as colony forming ability was greater in rec⁺ strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilites. In contrast, recA431 mutants were as sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA⁺ and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.     

Poly(ADP-ribose) metabolism appears normal in EM9, a mutagen-sensitive mutant of CHO cells

EM9 is a mutagen-sensitive CHO cell whose phenotype resembles that of normal CHO cells exposed to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis. This phenotype suggested that EM9 might be defective in poly(ADP-ribose) metabolism, but we now cannot find any abnormality in the synthesis or in the degradation of poly(ADP-ribose) in permeabilized EM9 cells. Thus the effects of 3-aminobenzamide on wild-type cells may be due to the inhibition of processes other than poly(ADP-ribose) synthesis. 3-Aminobenzamide enhances the cytotoxicity of EMS toward EM9 and control cells to the same degree.     

β-decay,Bremsstrahlen, and the origin of molecular chirality

Summary A brief review is presented of the Vester-Ulbricht -decay Bremsstrahlen hypothesis for the origin of optical activity, and of subsequent experiments designed to test it. Certain of our experiments along these lines, begun in 1974 and involving the irradiation of racemic and optically active amino acids in a 61.7 KCi⁹⁰Sr–⁹⁰Y Bremsstrahlen source, have now been completed and are described. After 10.89 years of irradiation with a total Bremsstrahlen dose of 2.5×10⁹ rads, crystallinedl-leucine, norleucine, and norvaline suffered 47.2, 33.6, and 27.4% radiolysis, respectively, but showed no evidence whatsoever of asymmetric degradation.d- andl-Leucine underwent about 48% radiolysis and showed 2.4–2.9% radioracemization. Other samples in solution were too severely degraded to analyze. Probable intrinsic reasons for the failure of the Vester-Ulbricht mechanism to afford asymmetric radiolysis in the present and related experiments involving -decay Bremsstrahlen are enumerated.A portion of this material was presented at the 7th International Conference on the Origins of Life, Mainz, FRG, July 10–15, 1983     

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.     

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

Changes in polyamines and related enzymes with loss of viability in rice seeds

Putrescine, spermidine and spermine of high vigour, low vigour and non-viable (classes 1, 2 and 3 respectively) seeds of Oryza sativa increased with loss of viability. The largest concentration of spermine was found in non-viable embryos. Spermine was absent in the husks of all the three categories of seeds. Arginine decarboxylase was greatest in high vigoured seeds and its activity gradually declined with loss of viability. However, diamine oxidase and polyamine oxidase activities gradually increased with the loss of viability of the seeds while DNA, RNA and protein contents decreased. The total content of polyamines increased on kinetin treatment but declined on ABA treatment. DNA, RNA and protein followed the same trend as polyamines. The polyamine contents increased by ca 3- and 4-fold, respectively, in high vigoured and low vigoured seeds on 10^?4 M kinetin treatment. The activity of ADC followed the same change as that of the polyamines in both cases, but the reverse was observed for the activities of diamine and polyamine oxidases.     

Chain length-dependent association of distamycin-type oligopeptides with A·T and G·C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC)·poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG·dC containing sequences at moderate ionic strength and are classified as highly dA·dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG·dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG·dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair--direct alkylation versus metabolic activation and breakdown. II. O,O-dimethyl-O-(1,2-dibromo-2,2-dichloroethyl)-phosphate and two O-ether derivatives of trichlorfon

The following organophosphates were tested for their ability to induce DNA damage in a rec-type repair test with Proteus mirabilis strains PG713 (rec- hcr-) and PG273 (wild-type) and point mutations in the his- strain TA100 of Salmonella typhimurium: O,O-dimethyl-O-(1,2-dibromo-2,2-dichloroethyl)-phosphate (NALED); trichlorfon-O-methyl ether (TCP-O-ME), O,O-dimethyl-(1-methoxy-2,2,2-trichlorethyl)-phosphonate; trichlorfon-O-methyl ether vinyl derivative (TCP-O-MEVD), O,O-dimethyl-(1-methoxy-2,2-dichlorovinyl)-phosphonate. All compounds were negative in the repair test but induced base pair substitutions in S. typhimurium. The mutagenicity of NALED is due to the direct alkylating ability of the parental molecule and to mutagenic metabolites generated by enzymatic splitting of the side chain. Glutathion-dependent enzymes in the S9-mix eliminate the mutagenic activity of NALED completely. Mutation induction by TCP-O-ME and TCP-O-MEVD is predominantly caused by the reactive O-methyl ether configuration of the side chain and is resistant to metabolic inactivation by NADPH- or glutathion-dependent enzymatic pathways in the S9-mix of mice.