Toxic effects of 2-methyl-4-dimethylaminoazobenzene in normal and partially hepatectomized rats

In order to obtain a more precise definition of the conditions under which 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) and liver cell proliferation play a role in the initiation of hepatocarcinogenesis, the toxicity of 2-Me-DAB for normal and partially hepatectomized rats was investigated. Continuous feeding of a basal low protein, low riboflavin diet supplemented with 2-Me-DAB was found to be highly toxic for male albino rats. All animals fed on such a diet died before 200 days. Sham operation and partial hepatectomy (PH) at 30 days of 2-Me-DAB feeding reduced the median survival time from 122 days to 107 and 94 days, respectively. Transfer to the basal diet after 30 days of 2-Me-DAB feeding and PH prolonged the median survival time to 216 days while 97% of the rats returned to the normal complete diet after the same treatments survived for more than 300 days. 2-Me-DAB was not necrogenic and there was no evidence of reparative proliferation or hepatic tumor formation in any group. Feeding rats with the 2-Me-DAB containing diet for 1 month delayed and strongly inhibited the mitotic response of the liver to the stimulus of partial hepatectomy. This is the result of a blockage of the cells in G1 as revealed by the fact that only 1% of the hepatocytes became labeled when 2-Me-DAB fed animals were injected with tritiated thymidine prior to sacrifice at 24 h post-hepatectomy, as compared to 40% in rats fed the normal or the control basal diet. This inhibitory effect of 2-Me-DAB is reversible however since rats returned to the normal diet for 1 or 2 months after 2-Me-DAB feeding showed percentages of mitoses and labeling indices comparable to those of control animals following PH. The number of abnormal mitoses was high (13%) in regenerating livers of rats fed 2-Me-DAB and the lesions responsible for this effect are apparently not repaired since 2-Me-DAB fed rats partially hepatectomized after being transferred to the normal diet for 1 or 2 months showed the same number of mitotic irregularities. The present results suggest that assays with 2-Me-DAB as 'pure initiator' or agent of selective toxicity should be pursued in attempts to improve existing experimental models of hepatocarcinogenesis.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Stimulation of DOPA Synthesis in the Superior Cervical Ganglion by Veratridine

We have investigated the effect of veratridine on DOPA (3,4-dihydroxyphenylalanine) accumulation by the superior cervical ganglion of the rat. Incubation of the ganglion with veratridine (50 microM) causes a 10-fold increase in the rate of DOPA accumulation. Veratridine-stimulated DOPA accumulation is blocked by tetrodotoxin, but not by cholinergic or adrenergic antagonists or by decentralization of the ganglion. The cyclic nucleotide 8-bromo cyclic GMP does not increase DOPA accumulation, and 8-bromo cyclic AMP causes only a 2-fold increase in DOPA accumulation, which is additive with the effect of veratridine. Thus, the action of veratridine appears to be independent of these cyclic nucleotides. The effect of veratridine on DOPA accumulation is probably due to a stable modification of tyrosine hydroxylase, since an increase in tyrosine hydroxylase activity can be measured in cell-free extracts of veratridine-treated ganglia. Both the increase in DOPA accumulation and the stable activation of tyrosine hydroxylase are dependent upon extracellular Ca2+. The activation of tyrosine hydroxylase by veratridine may be mediated by the depolarization of, and the subsequent entry of Ca2+ into, ganglionic neurons.     

Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.     

Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

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     

大鼠非致瘤四倍体细胞系的建立及细胞遗传学和酶学特征

从大鼠自然诱发的肉瘤细胞中,我们建立了一个四倍体细胞系(4n=84),命名为RC(ratcell)。它具有典型的成纤维细胞外形,能在玻璃表面贴壁生长,但不能生长在琼脂半固体培养基中。该细胞在含15％小牛血清的RPMI 1640培养基中生长良好,至今已连续繁殖112世代,细胞群体倍增时间约为15小时。染色体G-带分析表明,RC为整四倍体细胞,它的1条X染色体在第32至34区为均染区。RC细胞核仁组织者(NORs)活性显然比大鼠二倍体细胞NORs活性的加倍还高(P<0.001)。这个具有非常高NORs活性的RC细胞系对于研究细胞18S+28S rRNA基因转录活性的调控、基因表达与基因剂量关系有一定的意义。RC细胞还有异常高的磷酸酯酶活性,而且它的同工酶谱也与大鼠肌肉细胞明显不同。体内接种实验和扫描电镜的观察表明,RC是非致瘤细胞。RC细胞各号染色体的C-带图样与大鼠二倍体细胞无明显的差异。     

Influence of both coculture and BrdU on NOR activity of mouse,rat and human cells

Summary The coculture of mouse PG19 cells with human MGC cells can significantly suppress nucleolar organizer region (NORs) activity of both PG19 and MGC cells. 5-bormodeoxyuridine (BrdU) can also significantly suppress the NOR activity of rat RC cells, human MGC and Hela cells, and mouse PG19 cells: i.e. the average number of Ag-NORs and the number of chromosomes bearing Ag-NORs per cell decrease significantly. The degree of the suppression increases with increase in both BrdU concentration in the culture medium and BrdU treatment time. The suppressed NOR activity of the PG19 cells can gradually be restored when the BrdU-treated cells are transferred into BrdU-free medium for 50 h. In PG19 cells deoxycytidine (dC) can reverse the suppression of NOR activity caused by BrdU. Coculture plus BrdU treatment suppress the NOR activity of PG19 cells more severely than BrdU treatment alone. In coculture medium containing 30 g BrdU/ml, dC can also reverse the suppression of the NOR activity of PG19 cells but not that of the MGC cells. The degree of the reversion in the coculture plus BrdU treatment is significantly lower than that found with BrdU-treatment alone.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

Influence of dietary supplementation with thiamine on lead intoxication in rats

The influence of dietary supplementation with thiamine on lead (Pb) contents in blood and tissues, blood δ-aminolevulinic acid dehydratase (δ-ALAD) activity, and urinary excretion of δ-aminolevulinic acid (δ-ALA) was evaluated in male Sprague-Dawley rats. Groups of randomly selected animals were given a thiamine-deficient diet, a diet containing normal thiamine (20 mg/kg), or a thiamine-supplemented diet (50 mg/kg), along with control drinking water or water containing 100 ppm Pb, for 4 mo. Animals fed the thiamine-supplemented diet (50 mg/kg) and Pb showed decreased urinary excretion of δ-ALA and a decreased inhibition of δ-ALAD activity in blood compared to those given Pb with normal thiamine diet. The liver, kidney, and blood of rats receiving supplemental thiamine also contained significantly less Pb than the other two treatment groups given Pb-containing water. The protective effect of thiamine against Pb toxicity may be attributed to its interference with retention of the metal in body tissue, possibly resulting from the formation of excretable thiamine-lead complexes.