The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

Isolation and characterization of a carcinoma-associated antigen

GA733 is a murine IgG2a monoclonal antibody (MAb) against human gastric carcinoma and is highly tumoricidal in nude mice. The GA733 antigen is a cell surface protein with two subunits of 30,000 and 40,000 daltons. The antigen isolated by immunoaffinity chromatography consists mainly of the 30,000-dalton subunit which bears the GA733 epitope. This subunit displayed several isoelectric points between 6.9 and 7.7. Anti-colon carcinoma MAb 17-1A also detects this antigen, but probably binds to a different epitope.     

Proliferation dependence of topoisomerase II mediated drug action

Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.     

Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding

Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.     

Cytolytic activity of antigen-specific T cells with helper phenotype

We have investigated an unusual cytolytic activity displayed in vitro by cloned T cells which have the cell surface phenotype of helper T cells. When the cloned T cells are cultured with the appropriate antigen and antigen-presenting cells (APC), the T cells become activated in that they produce lymphokines and proliferate in an antigen-specific and major histocompatibility complex-restricted manner. At the same time, these T cells cause lysis of the APC. In addition, innocent non-histocompatible bystander cells present in the cultures can also be killed. The cytolytic activity may be involved in a mechanism of immune regulation.     

H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3' terminus and proceeds 3' to 5'

The relative decay of four human messenger RNAs, gamma globin, delta globin, c-myc and H4 histone, were compared in a cell-free system. Under appropriate conditions, they are degraded in vitro in approximately the same relative order as in vivo: histone faster than c-myc and delta globin faster than gamma globin. Degradation of polysome-associated H4 histone mRNA and of deproteinized histone mRNA begins at or near the 3' terminus. At least a portion of the mRNA then continues to be degraded in a 3' to 5' direction. Discrete 3'-terminal degradation hold-up points are observed, suggesting that 3' to 5' degradation occurs non-uniformly. Cycloheximide and puromycin inhibit protein synthesis but do not affect the rate or directionality of histone mRNA decay in vitro. We conclude that the rate-limiting step in H4 histone mRNA decay occurs at or near the 3' terminus and that at least a portion of the mRNA molecule is subsequently degraded 3' to 5', probably via a processive exonuclease.     

Respiration Rate of Steinernema feltiae Infective Juveniles at Several Constant Temperatures

Respiration was measured in dauer stages of the insect-parasitic nematode Steinernema feltiae (= Neoaplectana carpocapsae) at 7, 17, and 27 C. Respiration, Q₁₀, and nematode viability were temperature dependent. Mean O₂ consumption for 5 × 10⁵ nematodes the first 24 hours was 0.27 ml at 7 C, 0.83 ml at 17 C, and 2.68 ml at 27 C. The Q₁₀ was 3.10 for 7-17 C and 3.24 for 17-27 C. Some nematodes died during 2, 14, and 21 days at 27, 17, and 7 C, respectively. The respiratory quotient was below 1 at all temperatures tested. A standard asymptotic model is expressed as oxygen consumed = 2.77 * {1 - exponent[-time * exponent(-B + C * temperature)]}; where 2.77 is the maximum response at 27 C. This model estimates nematode O₂ consumption and viability at storage temperatures between 7 and 27 C. The nematodes died when the O₂ concentration reached 0.5 ml/5 × 10⁵ nematodes. This model may be used to predict O₂ requirements of S. feltiae infective juveniles when stored as a waterless concentrate.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Diurnal variations in plasma arginine vasotocin (AVT) concentrations in the ovine fetus

Previously we have demonstrated the presence of AVT in the blood of fetal sheep. The source is not clear, but AVT has been identified in fetal pineal and pituitary glands. In view of the circadian secretory pattern of the pineal gland, we questioned whether fetal plasma AVT levels might vary diurnally. Plasma samples from five chronically catheterized ovine maternal ewes and fetal lambs 129-135 days' gestation were obtained at 3-19 hourly intervals for 1-2 days (mean +/- S.E.M. = 35 +/- 6 hours. Plasma AVT levels were determined by radioimmunoassay. Results were analyzed by nonlinear curve fitting procedures to relate hormone levels with time of day. Plasma AVT values for maternal ewes did not vary during the day in response to light/dark periods. The curve for mean fetal plasma AVT plotted against time showed oscillations with a period of about 25 hours (p less than 0.05). Peak fetal AVT levels were observed at 1600 hours and minimal levels at 0400 hours. These results indicate that ovine fetal AVT secretion varies diurnally. The site of AVT secretion may be the pineal gland; however, confirmation of this and identification of the physiological stimuli for secretion of fetal plasma AVT require further information.