Cyrus P. Barnum

24-Hour rhythms at several levels of integration in mice on different lighting regimens.

Summary and conclusions The following variables were studied in the mouse under conditions standardized for evaluation of 24-hour periodicity: glucose and “corticosterone” in blood; mitoses in pinnal epidermis and hepatic parenchyma; liver glycogen and the hepatic metabolism of cytoplasmic phospholipid and ribonucleic acid and of nuclear deoxyribonucleic acid. For each variable, data obtained with light from 6 p.m. to 6 a.m. alternating with 12 hours of darkness, were compared with data obtained with light from 6 a.m. to 6 p.m. A shift in timing of 24-hour rhythms, following the inversion of lighting regimen came clearly and consistently to the fore. Ordinarily, environmental lighting is the dominant synchronizer of various 24-hour rhythmic functions, at different levels of organization of mice: by instituting an appropriate schedule of lighting and by allowing thereafter for the necessary shift time, one may set the peak or trough of rhythm to any chosen clock hour. Moreover, by controlling the timing of various periodic functions with respect to the environment, defined phase relations are brought about among the various physiologic rhythms themselves. The possibility to control timing of these rhythms by an easily manipulated environmental factor is of obvious practical interest, but it also constitutes a requisite for a basic analysis of functions, within the range in which they vary under ordinary circumstances.

Effect of hydroxyurea on regenerating rat liver

Abstract Hydroxyurea has been shown to be a potent inhibitor of DNA synthesis in regenerating rat liver with no measurable effect of RNA synthesis. No detectable difference was noted in incorporation of 14 C-glycine into cytoplasmic protein or into acid-insoluble nuclear protein, but the incorporation into nuclear histone was only 61% of the control value, suggesting that hydroxyurea may inhibit the synthesis of histone in addition to, or as a consequence of, its effect on DNA.

Quantitative Distribution of an Esterase Among Cytoplasmic Components of Mouse Liver Cells.

Summary The esterase in the cytoplasm of mouse liver cells was found to be localized predominantly in the microsome fraction which contained an average of 47% of the enzyme in the whole tissue and 63% of that in the entire cytoplasm. Other cell fractions, the nuclear material, the large cytoplasmic granules, and the supernate from the microsome separation contained an average of 17%, 17%, and 14%, respectively, of the enzyme in the whole tissue. Enzyme activity per fig of N was greatest in the microsomes which contained a concentration that was 4.2 times that of the whole tissue. In the other particulate fractions, the nuclear material, the large cytoplasmic granules, and the supernate from the microsome separation, the enzyme concentration was found to be 0.6, 1.6, and 0.3 times, respectively, that of the whole tissue. The “lipoprotein” fraction of the microsomes displayed about 3 times the enzyme concentration found in the “nu-eleoprotein” fraction. Summation of the enzyme content of the substituent fractions indicated no appreciable loss during the separation procedures. We are indebted to Dr. J. J. Bittner for the mice used in these experiments.

Partition of cytoplasmic lipides.

Abstract By means of differential centrifugation three fractions: large granules (L 2 ), microsomes (M 1 ), and supernatant fluid (MS), have been isolated from the cytoplasm of mouse liver cells and the contained lipides have been extracted and characterized. The particulate fractions, large granules and microsomes, make up approximately 38% of the total solids of the cytoplasm, but they contain 62% of the total lipide and 85% of the phospholipide. The phospholipide of the particulates has an N:P ratio of 1 while the supernatant phospholipide has an N:P ratio of 1.3, suggesting that the supernatant fluid contains lipides of higher nitrogen content than that of either lecithins or cephalins. The fatty acids of the large granule fraction are highly unsaturated and contain 20% of fatty acids with four double bonds. The unsaturation of the fatty acids of microsomes and the supernatant fluid is comparable, as indicated by the iodine values, but the supernatant fluid contains much more of the fatty acids with one double bond and less of the fatty acids with four double bonds than do the microsomes.

THE EFFECT OF DIISOPROPYLPHOSPHORO-FLUORIDATE (DFP) ON MOUSE BRAIN PHOSPHORUS METABOLISM

THE pharmacological effects of diisopropylphosphorofluoridate (DFP)? are commonly attributed to its inhibition of nervous system cholinesterases. There are, however, a number of indications that other factors may be involved (MCNAMARA et a[., 1454). Following inhibition by 32P-labelled DFP, cholinesterase and other enzymes such as chymotrypsin are found on hydrolysis to yield ~2P]-phosph.oserine; this suggests that the reaction of DFP with serine residues prevents the catalytic action of these enzymes (SCHAFFER et a]., 1953, 1954). The possibility that serine may also play an important role in enzymes of phosphorus metabolism is indicated by the relatively rapid rate at which protein-bound phosphoserine of animal tissues becomes labelled following the injection of 32P-labelled orthophosphate (KENNEDY and SMITH, 1954). On the basis of these considerations it seemed plausible that DFP might interfere with phosphorus metabolism by tying up serine groups which may be necessary for the functioning of the enzymes involved. Thus, KENNEDY and KOSHLAND (1957) have observed that serine may be involved in the action of phosphoglucomutase, and that this enzyme could be inhibited by DFP. Such an effect of DFP on the phosphorus metabolism of the nervous system could conceivably be responsible for some of the pharmacological effects of this agent.

Metabolic activity and intracellular distribution of nucleic acid phosphorus in regenerating liver

Abstract The specific activity-time curves for the various liver nucleic acids were determined within a 6-hr. period from 58 to 64 hr. after partial hepatectomy in mice using P 32 as the tracer. The nuclear, supernatant, ultramicrosomal, and microsomal RNAs are listed in the order of decreasing specific activities. The protein and phospholipide phosphorus specific activities increased rapidly during the first hour, and all, with the exception of those from nuclei that were lower, were almost equal to the IP specific activity 6 hr. after P 32 injection. A study of the quantitative distribution of RNA revealed that about 40% of the cell RNA in the 60-hr. regenerating liver is associated with the microsome fraction. Only about 5–10% could be accounted for by each of the RNAs of the other fractions. Assuming the condition of the steady state and metabolic homogeneity of the various nucleic acid phosphorus pools, equations representing different metabolic schemes were tested. It was shown that the data were in reasonable agreement with a metabolic scheme according to which M-RNA is formed from N-RNA at the rate of 3.4%/hr., and N-RNA appears at the rate of 24 and 34%/hr. from IP and M-RNA, respectively. Limiting fractional rates for the appearance of the various RNAs were calculated using the liver inorganic phosphorus specific activity-time curve in place of that of the immediate precursor; these were 1–2% and 25%/hr. for cytoplasmic and nuclear RNA, respectively. This fractional renewal rate for N-RNA is much greater than most values reported in the literature and, even so, must be considered a limiting minimum value. It seemed unjustified to assume the steady-state condition during the kinetic study for the rates of reactions leading to DNA on the basis of the shape of its specific activity-time curve and on the morning-afternoon differences in the 1-hr. relative specific activity. Therefore, metabolic reactions involving DNA were not tested. An equation was developed to test whether the U fraction is a mixture of particles containing M- and S-RNA. The calculated specific activities from approximately a 50:50 mixture of S- and M-RNA were in fairly good agreement with the observed values of U-RNA.

Chemistry of the Liver Cytoplasm of Normal, Fasted and Cirrhotic Mice.

Summary and discussion Chemical changes in the cytoplasm of the mouse liver cell after a short fast and at cirrhosis are reported. These changes occur most dramatically in the lipide fraction during a 24 hour fast resulting in an increase of 3 to 4 fold above normal. This lipide is non-phospholipide in character. Fasted cirrhotic liver differs from the fasted normal liver cytoplasm in that the lipide does not increase so markedly and the phospholipide decreases to about 60% of the normal fasted value. The iodine values of the normal-fasted and the cirrhotic-fasted liver cytoplasm fatty acids decrease below that of the normal values. In addition to a lipide increase in the fasted-cirrhotic liver cytoplasm there is also a decrease of “ribonucleic acid” and phospholipide in one gram wet weight of the ground fasted cirrhotic liver. It is possible that there is an interchange of phospholipide during a fast as evidenced by the lipide nitrogen to phosphorus ratio. This interchange would consist of substituting normal phospholipide with a lipide or phospholipide which contains a great deal of nitrogen.