Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

缺乏抗坏血酸对豚鼠胆固醇代谢的影响

边缘性缺乏抗坏血酸之豚鼠,于三周内其肝脏及小肠粘膜3-羟-3-甲基戊二酰辅酶A还原酶(HMGR)活力均下降到原有水平的50％,但肝脏胆固醇7α-羟化酶活力尚无显著性改变。坏血病豚鼠(三周内)上述几种酶活力都下降至原有水平的50％左右。豚鼠摄取抗坏血酸不足,其血清总胆固醇浓度显著增加,而血清高密度脂蛋自胆固醇浓度显著减少,其改变程度与抗坏血酸缺乏状况一致。     

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.     

Macrophage mediation of the inhibitory effects of elevated intracellular levels of adenosine-3':5' cyclic monophosphate (cAMP) on macrophage-Trypanosoma cruzi association

Presence of theophylline and dibutyryl-cAMP—agents which cause elevation of intracellular levels of cAMP—in in vitro systems in which murine macrophages interact with virulent blood forms of Trypanosoma cruzi resulted in a marked inhibition of cell-parasite association (i.e., decreased surface binding and/or internalization). This effect was evidenced in terms of significant reductions in both the percentage of infected macrophages and the average number of trypanosomes per 100 macrophages. Pretreatment of the macrophages with these agents produced a similar inhibition whereas pretreatment of the parasites had no significant consequence on the interaction. The inhibitory effect was transient since it was no longer seen after 30 min of incubation of the treated macrophages in fresh medium. Thus, the inhibitory effect is exerted through a transient effect on the macrophage.     

Regulation of Na/K/Cl cotransport in vascular smooth muscle cells

The regulation of Na/K/Cl cotransport was investigated in vascular smooth muscle cells. That a Na/K/Cl cotransport system exists was established by the finding that the ouabain insensitive K influx was sensitive to the "loop" diuretic bumetanide. Furthermore, bumetanide sensitive K influx was dependent upon the presence of both Na and Cl in the extracellular milieu. Bumetanide sensitive K influx was inhibited by agents which elevate cellular cyclic AMP levels, and to a lesser extent by agents which elevate cellular cyclic GMP levels. When serum, EGF or TPA was added, bumetanide sensitive K influx was enhanced. These results suggest that vascular smooth muscle cells have a ouabain insensitive, bumetanide sensitive Na/K/Cl cotransport system which is stimulated by serum, EGF or TPA and inhibited by cAMP or cGMP.     

Identification of a tumor inhibitory factor in rat ascites fluid

A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.     

Phosphorylation of zymogen granule membrane proteins in intact rat pancreatic acinar cells

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.     

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.     

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.