The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N⁴-bis(2-carboxyethyl)deoxycytidine (3,N⁴-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N⁴-BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3,N⁴-bis(2-carboxyethyl)cytosine (3,N⁴-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d₂₇) tri-TMS derivative of 3-CEC. The structure of 3,N⁴-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N⁴-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N⁴-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H₂O at 37°C and pH 7.0–7.5.     

Macrophage-hybridomas: generation, structure, and function

We report the generation of macrophage-hybridomas, obtained by somatic cell fusion between macrophage-enriched C3H.eB spleen cell population, and a drug-resistant MPC-11 myeloma cell line, designated as 4T00.1L1 clone. Screening for hybridomas possessing macrophage properties was carried out by assaying the presence of two macrophage-specific enzymes: lysozyme and nonspecific esterase. Two hybridomas, E2-7 and E2-10, were selected for further studies. We found that clones of E2-7 (E2-7.7) did not express Fc receptors but possessed cell-surface Ia molecules. In contrast, clones of E2-10 (E2-10.20) possessed Fc receptors but were devoid of Ia molecules. E2-7.7 did, however, express Fc receptors after mitomycin treatment, whereas E2-10.20 eliminated the expression of Fc receptors after treatment with mitomycin C. Opsonized erythrocytes were phagocytized by E2-10.20 cells, but not by E2-7.7. Phagocytosis was thus correlated with the possession of Fc receptors. Testing the response of KLH-primed lymph node cells to KLH-pulsed hybridoma cells, we found that E2-7.7 cells caused antigen-specific lymphoproliferative response, whereas E2-10.20 did not. Thus, antigens could be presented by E2-7.7 but not by E2-10.20 cells. The response was shown to be mediated by T but not by B lymphocytes. The difference in antigen-presenting capacity could not be attributed to differences in antigen uptake by the different hybridomas, because the two hybridomas manifested the same level of pinocytosis. Both hybridomas produced IL1. The differences in the properties of the two hybridomas may indicate that the normal partners represent two distinct subpopulations of macrophages. The segregation of functional properties among the hybridoma clones may lead to a clarification of the dependence of distinct functions on defined molecular structures.     

Statistical analysis of multipath neural systems

Peripheral sensory and motor systems may be characterized by models consisting of multiple parallel convergent pathways, each described by the same set of equations, but having different parameter values in each path. Such models, although deterministic, are best analyzed using a statistical approach, which is illustrated here by analysis of several simple multipath models composed of linear dynamic elements and static non-linear elements. Relationships between instantaneous means of signals at different points in such systems are used to show that a multipath system can exhibit behaviour which would not be expected from observations of individual pathways. Mechanisms for linearization of static non-linearities are briefly described. Important implications for neurophysiologists are discussed.     

Purification and physiological role of a peptide stabilizing factor of rat liver phosphofructokinase.

A substance has been purified to apparent homogeneity from rat liver which, as previously reported (Dunaway, G. A., Jr., and Segal, H. L. (1974) Biochem. Biophys. Res. Commun. 56, 689-696), specifically stabilizes the major liver isozyme of phosphofructokinase (PFK-L2) against thermal inactivation and whose level in vivo changes in parallel with and in precedence to that of the enzyme. Molecular weight determinations gave values around 3,500. Evidence for the peptide nature of the factor includes its correspondence with ninhydrin-positive material on gel filtration and paper electrophoresis and its susceptibility to pronase. Electrophoretic behavior indicated at least one free amino group and several carboxyl groups. Amino acid analysis of the peptide yielded only glutamate, glycine, and half-cystine, in equimolar amounts. However, neither GSH nor GSSG have PFK-L2-stabilizing activity. No free sulfhydryl groups were present. Chemical analysis for tryptophan was also negative. The ultraviolet spectrum confirmed the absence of aromatic amino acids. The spectrum exhibited a characteristic peptide peak at 190 nm with no absorbance beyond 240 nm. The factor is unstable to storage in the cold except in the presence of glucose or dithiothreitol. Sucrose, fructose, and GSH were ineffective in this regard. It was slowly denatured by heat or reduced pH even in the presence of glucose. The factor was induced in fasted animals specifically by glucose, of the nutrients tested, and in diabetic animals by insulin. Induction by both glucose and insulin was blocked by cycloheximide and actinomycin. The time course of the glucose induction was the more rapid of the two with a marked overshoot to 3 times normal levels at 12 hours. Increased levels of the factor preceded the increased levels of PFK-L2 brought about by glucose or insulin administration. Native PFK-L2 was inactivated by lysosomal extracts, and this inactivation was strongly inhibited by the peptide factor. These results are in accord with the proposal that the peptide plays a role in regulating PFK-L2 turnover in vivo. The factor also activated the phosphofructokinase-catalyzed reaction by promoting fructose-6-P binding. This effect is analogous to that of AMP on the kinetics of the reaction; however, the factor effect was additive to that of AMP, and the factor did not reverse inhibition by excess ATP as does AMP. We postulate that the stabilizing factor affects an equilibrium between PFK-L2 conformers in favor of one more resistant to lysosomal and thermal inactivation and with greater affinity for fructose-6-P.     

Effect of a cyclic enkephalin analog on the protein content in the hippocampal neurons of rats during the acquisition of a 2-way avoidance conditioned reflex

The protein content has been determined by means of cytointerferometry in neurons of fields CA-1 and CA-3 of the dorsal hippocampus in rats, which were trained in a conditioned reflex of two-way avoidance (CRTA) with the action of subcutaneously injected enkephalin cyclic analogue (ECA) in a dose 10 mkg. It has been found that after ECA injection the protein content in the neuronal nuclei of the hippocampal CA-3 field reduces. The acceleration of the CRTA elaboration occurring during the action of ECA is accompanied by a drastic increase of the protein content in the neuronal nuclei of the CA-3 field. The ECA administration to the rats of the active control groups to which were presented the same number of unpaired conditioned and unconditioned stimuli as during the CRTA elaboration also enhances the protein content in the neurons of the CA-3 field. The rats of all investigated groups in the neurons of the CA-1 field display no such significant shifts. The conclusion has been drawn that ECA produces a regulating influence on protein metabolism in hippocampal neurons depending on their functional state.     

Biphasic stimulation of cellular calcium concentration by 3,5,3'-triiodothyronine in rat thymocytes

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.