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Adenylate cyclase [atp pyrophosphate-lyase (cyclizing): EC 4.6.4.4] activities were examined in mouse LM cell (fibroblast) membranes that were supplemented with ethanolamine and/or fatty acids. The supplements were incorporated into the plasma membrane phospholipids in significant amounts. Fatty acid supplementations had distinct effects as compared to polar head group supplementations. All lipid supplementations increased basal adenylate cyclase activity relative to control cells grown in choline-containing medium. Double supplementation with ethanolamine and linoleate increased the specific activity of adenylate cyclase up to 4-fold. Activity in the presence of fluoride was unaffected by ethanolamine supplementation, but was increased by fatty acid supplementation. In contrast, prostaglandin E1 stimulation was 4.2-fold in controls and ethanolamine and/or elaidate supplements, 6-fold in choline plus linoleate supplements, and 3.1-fold in ethanolamine plus linoleate supplements. Differences in activity could not be ascribed to changes in membrane protein composition in supplemented cells, and could be abolished by detergent solublization. Fluidity of the supplemented membranes was monitored by fluorescence polarization, and no correlation was observed between membrane viscosity and adenylate cyclase activity or hormone stimulation. These results emphasize the importance of the membrane lipid phase for this enzyme.

Of the molecular biochemical alterations that occur during apoptosis, activation of caspases, notably caspase-3, is probably the most attractive for developing specific in vivo molecular imaging probes. We recently designed a library of isatin-5 sulfonamides and selected [18F]ICMT-11 for further evaluation on the basis of subnanomolar affinity for activated capsase-3, high metabolic stability, and facile radiolabeling. In this present study, we have demonstrated that [18F]ICMT-11 binds to a range of drug-induced apoptotic cancer cells in vitro and to 38C13 murine lymphoma xenografts in vivo by up to 2-fold at 24 h posttreatment compared to vehicle treatment. We further demonstrated that the increased signal intensity in tumors after drug treatment, detected by whole body in vivo microPET imaging, was associated with increased apoptosis. In summary, we have characterized [18F]ICMT-11 as a caspase-3/7 specific PET imaging radiotracer for the assessment of tumor apoptosis that could find utility in anticancer drug development and the monitoring of early responses to therapy.

Dexamethasone and progesterone have been found to accelerate the time of initiation and enhance the rate of myelin synthesis in Schwann cell/neuronal cocultures. The expression of mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3beta-hydroxysteroid dehydrogenase (converts pregnenolone to progesterone), and the progesterone receptor were detected and markedly induced during peak myelin formation in the cocultures. The mRNA for the glucocorticoid receptor was detected, but was found to be constituitively expressed. In addition, the specific activity of 3beta-hydroxysteroid dehydrogenase was measured and found to increase by 10-fold. The mRNA for cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase also were found to be induced during the differentiation of O-2A precursor cells to oligodendrocytes. Fibroblast growth factor and platelet-derived growth factor were found to have proliferative effects on Schwann cells, but they had no effect on the initiation or the rate of myelin formation. These results demonstrate that myelin-forming cells have inducible enzymes responsible for steroid biosynthesis and suggest a critical role for endogenous steroid hormones in signaling the initiation and enhancing the rate of myelin formation.

Methods have been devised to significantly alter the phospholipid composition of LM cells grown in serum-free tissue culture medium. The polar head groups of the phospholipids, as well as the acyl groups of these lipids, can be changed. When LM cells were grown in medium containing choline analogues, either N-methyl-ethanolamine or N,N-dimethylethanolamine, or the unnatural analogues, 1-2-amino-1-butanol or 3-amino-1-propanol, up to 50% of the cellular phospholipids contained the analogue supplied. When linoleate was added to the cells as a bovine serum albumin complex, up to 40% of the fatty acids of the phospholipids were linoleate. Under the conditions discussed, the polar head group composition, the fatty acid composition, or both together could be varied in the membrane phospholipids.

The reversible binding of the haptens 2,4-dinitrophenyllysine (DNP-lysine) and 2,4,6-trinitrophenyllysine (TNP-lysine) to either the nitrophenyl-binding myeloma protein MOPC-315 or to pooled mouse anti-DNP or anti-TNP antibodies produces large and characteristic extrinsic Cotton effects (in the circular dichroic spectra). Despite the similarities in binding characteristics of the three proteins, the circular dichroic spectra produced by the haptens bound to the active sites of these proteins were markedly different. Extrinsic Cotton effects, therefore, provide a powerful new probe of the structure of the reversible complex formed between a hapten and an antibody active site.

Nucleic acid and protein synthesis were studied in temperature-sensitive mutants defective in phospholipid synthesis. The defect is due to a single mutation in glycerol 3-phosphate acyltransferase (EC 2.3.1.15). The results show that at the restrictive temperature not only does phospholipid synthesis cease, but DNA, RNA, and protein synthesis also cease. Active transport continues, however, indicating that the cells do not become leaky or lose their energy supply. These results suggest that phospholipid synthesis is coupled to DNA, RNA, and protein synthesis.

Two homogeneous univalent hapten-protein conjugates, prepared by the covalent attachment of a single 2,4-dinitrophenyl (DNP-) or 2,4,6-trinitrophenyl (TNP-) side chain to the cysteine-SH in the active site of the enzyme papain, have been found to exhibit large Cotton effects in the wavelength region of the absorption bands of the DNP or TNP groups. This indicates that the DNP or TNP groups are noncovalently bound to some asymmetric region(s) of the papain molecule. These homogeneous papain conjugates are antigens that can elicit anti-DNP or anti-TNP antibody production in mice or rabbits. It is suggested that the noncovalent binding of a hapten to the surface of its carrier protein may profoundly affect the characteristics of the anti-hapten antibodies that are elicited. It has also been observed that the specific binding of these papain conjugates, and of the simple haptens DNP-lysine or TNP-lysine, to anti-hapten antibodies produces characteristic extrinsic Cotton effects.

The effects of temperature and of the action of a purified phospholipase C enzyme preparation on human red blood cell membranes has been investigated by chemical analyses, circular dichroism, and proton magnetic resonance measurements. The results indicate that a substantial fraction of the phospholipids and the proteins of the membranes can change structure independently of one another, suggesting a mosaic pattern for the organization of the lipids and proteins in membranes.