Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH)) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.     

On the distribution of axonal terminals containing spheroidal and flattened synaptic vesicles in the hippocampus and dentate gyrus of the rat and cat

Summary A quantitative analysis has been made of the distribution of presynaptic profiles containing round (or spheroidal) and flattened (or ellipsoidal) synaptic vesicles in the apical and basal dendritic zones and in the layer of pyramidal cell somata of fields CA₁ and CA₃ of the hippocampus, and in the molecular and granular layers of the dentate gyrus of the rat and cat.In the apical and basal dendritic zones of fields CA₁ and CA₃ the overwhelming majority of the synapses are of the asymmetrical variety, the axon terminals ending principally upon dendritic spines, and to a lesser extent upon the shafts and secondary or tertiary branches of the dendrites. Between 1 and 8% of the axon terminals in these zones contained flattened vesicles: all of these formed symmetrical contacts upon medium-sized or large dendritic shafts. In the molecular layer of the dentate gyrus a slightly higher percentage of flattened vesicle containing profiles was observed (10%); again these formed symmetrical contacts upon dendritic shafts. In the stratum pyramidale of the hippocampal fields and the stratum granulosum of the dentate gyrus of the rat, flattened vesicle containing synapses are two or three times more numerous than those with spheroidal vesicles. In the cat hippocampus the axosomatic synapses are about equally distributed between those containing round, and those with flattened vesicles.The finding that at the focus of post-synaptic inhibition, at the level of the pyramidal cell somata, the majority of the axon terminals contains flattened synaptic vesicles, whereas in the region of termination of the extrinsic, commissural and long association pathways (all of which are excitatory) virtually all the synapses contain round vesicles, strongly supports the view that endings containing flattened vesicles mediate post-synaptic inhibition in the hippocampal formation.Supported in part by Grant EY-00599 from the National Eye Institute.We should like to thank Mr. Paul Myers and Mr. Milburn W. Rhoades for their technical assistance, and Mrs. Doris Stevenson for secretarial help.     

Investigation of the Effect of Some Optically Active Imine Compounds on the Enzyme Activities of hCA‐I and hCA‐II under In Vitro Conditions: An Experimental and Theoretical Study

Inhibitors of carbonic anhydrase (hCA; EC 4.2.1.1) are used as medicines for many diseases. Therefore, they are very important. In this study, a known series of Schiff bases were synthesized and their effects on the activities of hCA‐I and hCA‐II, which are cytosolic isoenzymes of carbonic anhydrase, were investigated under in vitro conditions. The synthesized compounds (H1, H2, H3, and H4) were found to cause inhibition on enzyme activities of hCA‐1 and hCA‐II. IC₅₀ values of H1, H2, H3, and H4 compounds were 140, 88, 201, and 271 μM for hCA‐I enzyme activity and 134, 251, 79, and 604 μM for hCA‐II enzyme activity, respectively. The synthesized Schiff bases were characterized by several methods, including ¹H NMR, FT‐IR, elemental analysis, and polarimetric measurements. Correlation coefficient square values (R²) of comparison of the theoretical and experimental ¹H NMR shifts for H1, H2, H3, and H4 compounds were found as 0.9781, 0.9814, 0.9758, and 0.8635, respectively.     

Novel dimeric bis(7)-tacrine proton-dependently inhibits NMDA-activated currents

Bis(7)-tacrine has been shown to prevent glutamate-induced neuronal apoptosis by blocking NMDA receptors. However, the characteristics of the inhibition have not been fully elucidated. In this study, we further characterize the features of bis(7)-tacrine inhibition of NMDA-activated current in cultured rat hippocampal neurons. The results show that with the increase of extracellular pH, the inhibitory effect decreases dramatically. At pH 8.0, the concentration-response curve of bis(7)-tacrine is shifted rightwards with the IC(50) value increased from 0.19+/-0.03 microM to 0.41+/-0.04 microM. In addition, bis(7)-tacrine shifts the proton inhibition curve rightwards. Furthermore, the inhibitory effect of bis(7)-tacrine is not altered by the presence of the NMDA receptor proton sensor shield spermidine. These results indicate that bis(7)-tacrine inhibits NMDA-activated current in a pH-dependent manner by sensitizing NMDA receptors to proton inhibition, rendering it potentially beneficial therapeutic effects under acidic conditions associated with stroke and ischemia.     

Toxicity of Xanthene Dyes to Entomopathogenic Fungi

Effects of xanthene dyes on mycelial growth and conidial germination in three species of entomopathogenic fungi, Beauveria bassiana, Metarhizium anispoliae and Paecilomyces fumosoroseus, were evaluated in a variety of assay systems. In a disk-diffusion assay, erythrosin B and phloxine B (but not eosin B) produced zones of inhibition in colonies of all three species under continuous exposure to light at disk-loadings of 100mug. None of the dyes produced zones of inhibition in the absence of light at disk loadings of 100mug. Both erythrosin B and phloxine B inhibited mycelial growth of all three species in the light in a dose-dependent manner. Weaker dose-responses for inhibition of growth in the dark were observed for some fungus/dye combinations. Erythrosin B, tested singly, completely inhibited conidial germination in the light in all eight fungal strains tested at 100mug ml-1 medium, but failed to inhibit conidial germination in any of these strains in the dark at the same concentration of dye. For single strains of each of the three fungi, erythrosin B and phloxine B inhibited conidial germination in a dose-dependent manner in the light with IC50s < 6.2mug dye ml-1 medium for all fungus/dye combinations. Phloxine B was a more potent inhibitor of germination than erythrosin B for all three fungal species. At fixed dosages of erythrosin B and phloxine B, inhibition of conidial germination in all three species increased with time of exposure to light. These results constitute the first quantitative demonstration of photodynamic inhibition of conidial germination in fungi by xanthene dyes.     

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K _iE and K _iES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO. Received: 3 November 1998 / Accepted: 1 March 1999     

Evaluation of the herbicidal potential of some fungal species against Bidens pilosa,the coffee farming weeds

Weeds are the most productive limiting factor, especially in organic farming systems where the uses of synthetic herbicides are not allowed due to their negative impacts. Hence, synthetic herbicides need to be replaced with biological herbicides for weed management. Thus, the present study was designed to evaluate the herbicidal activity of conidia suspensions from Aspergillus niger, Trichoderma asperlium, Trichoderma atroviride, Trichoderma hamatum, Trichoderma harzanium, Trichoderma longibrachatum and Trichoderma viride against Bidens pilosa weed via a series of laboratory and lath-house conditions that laid out in a CRD and RCBD, respectively, with three replications for each bioassay. The results revealed that all fungi, except T. longibrachatum, had significantly reduced seed germination as well as early growth of the target weed compared to the untreated control. The inhibitory effects were measured to be varied among the types of conidia suspensions of fungal species and their level of concentration. The highest rate of inhibition was observed for conidia suspension from A. niger which suppressed with the maximum seed germination inhibitory level (65%) over control. Likewise, the plumule and radicle growth length of the target weed also significantly inhibited by the tested fungi (ranging from 10 to 85% and 34 to 97%) compared to the control, respectively. Based on their efficacy in the laboratory bioassay, the herbicidal potential of selected fungi was further evaluated in pot experiments. In contrarily to laboratory observations, the effect of different fungal conidia suspensions on various growth parameters of the targeted weed was insignificant in the lath-house experiments. In conclusion, the application of A. niger displayed some potential green light to be investigated as a biocontrol agent with promising retarding in the germination and early growth of B. pilosa. Hence, we recommend further investigation of those fungi under field conditions on different coffee weed species.