Nonphotic phase shifting in female Syrian hamsters: interactions with the estrous cycle

Nonphotic phase shifting of circadian rhythms was examined in female Syrian hamsters. Animals were stimulated at zeitgeber time 4.5 by either placing them in a novel running wheel or by transferring them to a clean home cage. Placement in a clean home cage was more effective than novel wheel treatment in stimulating large (> 1.5 h) phase shifts. Peak phase shifts (ca. 3.5 h) and the percentage of females showing large phase shifts were comparable to those found in male hamsters stimulated with novel wheels. The amount of activity induced by nonphotic stimulation and the amount of phase shifting varied slightly with respect to the 4-day estrous cycle. Animals tended to run less and shift less on the day of estrus. Nonphotic stimulation on proestrus often resulted in a 1-day delay of the estrous cycle reflected in animals' postovulatory vaginal discharge and the expression of sexual receptivity (lordosis). This delay of the estrous cycle was associated with large phase advances and high activity. These results extend the generality of nonphotic phase shifting to females for the first time and raise the possibility that resetting of circadian rhythms can induce changes in the estrous cycle.     

Phase shifting the hamster circadian clock by 15-minute dark pulses

The mammalian circadian pacemaker can be phase shifted by exposure to a period of darkness interrupting otherwise continuous light. Circadian phase shifting by dark pulses was interpreted originally as reflecting a photic mirror-image mechanism, but more recent observations suggest that dark pulse-induced phase shifting may be mediated by a nonphotic, behavioral state-dependent mechanism. The authors recently presented evidence indicating that the dark-pulse phase response curve (PRC) is in fact a complex function, reflecting both photic mirror image and nonphotic mechanisms at different phases of the circadian cycle. Previous studies of dark pulse-induced phase shifting have universally employed relatively long (2 to 6 h) pulses, which complicates PRC analysis due to the extended segment of the underlying PRC spanned by such a long pulse. The present study was therefore designed to examine the phase-shifting effects of brief 15-min dark pulses presented at both mid-subjective day and subjective dusk, and to explore the possible activity dependence of these effects by using physical restraint to prevent evoked locomotor activity. The results indicate that 15-min dark pulses are effective phase-shifting stimuli at both midday and dusk. Furthermore, as with longer dark pulses, phase shifting by 15-min dark pulses is completely blocked by physical restraint during subjective day but combines in a simple additive manner with the independent phase-shifting effect of restraint at subjective dusk.     

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.     

Are protein synthesis inhibition and phase shifting of the circadian clock in Gonyaulax correlated?

We describe a method whereby the effect of protein synthesis inhibitors upon protein synthesis in Gonyaulax cultures may be reliably measured. Using this method, we found that protein synthesis inhibition and clock resetting were correlated, but that the correlation was not as close as has been reported in other systems. The effect of the inhibitors anisomycin and cycloheximide upon phase shifting of the circadian clock was a function of the illumination and temperature conditions to which the cells were subjected, but these factors did not appear to influence the inhibition of protein synthesis by these drugs. Cellular protein synthesis did not recover immediately from the inhibitors' effects; depending upon the previous concentration of the inhibitor, translational recovery from the drugs may require hours. This observation has important implications for the analysis of any phase response curve when the stimulus is a chemical.     

Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature

We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41 degrees C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of alpha 2-macroglobulin, compartmentalization of newly synthesized acid hydrolases, resistance to ricin, and sensitivity to diphtheria and Pseudomonas toxins and modeccin. The three mutants also displayed decreased sialylation of some secreted glycoproteins at 41 degrees C, reminiscent of the nonconditional mutant DTG1-5-4 that showed both endocytic and Golgi- associated defects (Robbins, A.R., C. Oliver, J.L. Bateman, S.S. Krag, C.J. Galloway, and I. Mellman, 1984, J. Cell Biol., 99:1296-1308). Phenotypic changes were detectable within 30 min after transfer of the mutants to 41 degrees C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39 degrees C, the mutants exhibited many but not all of the changes manifested at 41 degrees C; resistance to diphtheria and Pseudomonas toxins required the higher temperature. Analysis of cell hybrids showed that B3853 and DTG1- 5-4 are in one complementation group ("End1"); M311 and I223 are in another ("End2"). In the End1 mutants, loss of endocytosis correlated with complete loss of ATP-dependent endosomal acidification in vitro; in the End 2 mutants partial loss of acidification was observed. At the nonpermissive temperature, residual levels of endocytic activity in B3853 and M311 were nearly identical; thus, we conclude that the differences measured in endosomal acidification in vitro reflect the different genetic loci affected, rather than the relative severity of the genetic lesions. The mutations in M311 and I223 appear to have different effects on the same protein; in I223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis.     

Light-induced phase shifting of the circadian clock in Neurospora crassa requires ammonium salts at high pH

Effects of external ionic conditions on light induced phase shifting of the circadian rhythm of conidiation in Neurospora crassa were examined in simple buffer solutions for discerning effects of individual ions. Mycelia were cultured to liquid media of different pHs and then transferted to 10 mM piperazine-N,N-bis(2-ethanesulmonic acid) (Pipes) buffer of various pHs and irradiated with while light. The phase of the rhythm of dark controls was not changed by transfer from medium to buffer. When mycelia were cultured in media of pH above 6.7, light did not advance the phase of the clock in Pipes buffer alone. However, light-induced phase advance was restored when an ammonium salt was added to buffer of pH higher than 7.6. An amination-defective mutant, bd am, showed the same response to ammonium nitrate as the wild-type strain, bd. Ammonium must be present before light irradiation for restoration of phase shifting. Free-amino-acid pools in the cells were changed by treatment with Pipes buffer: aspartle acid, glutamic acid, ammonia, glutamine and ornithine levels decreased, while lysine and histidine increased. Addition of ammonium nitrate to Pipes buffer resulted in further changes in amino-acid pools; lysine, histidine, arginine, alanine and ornithine decreased, and glutamine levels increased. Irradiation did not result in significant changes in amino acid pools.Abbreviation Pipes piperazine-N,N-bis(2-ethanesulfoniccid)     

Respiration-deficient Chinese hamster cell mutants: biochemical characterization.

We have previously classified 35 of our respiration-deficient mutants into seven complementation groups and one "overlapping" mutant which does not complement mutants from groups I and II. In this paper we report on the biochemical characterization of representatives of complementation groups I, II, VII, and the "overlapping" mutant. We show that these mutants all have a defect in complex I of the electron-transport chain. The general features of these mutants are: (1) a low rate of O2 consumption in whole cells; (2) a low rate of release of 14CO2 from [2-14C] pyruvate, [1-14C] pyruvate, and [3-14C] beta-hydroxybutyrate; (3) a low rate of release of 14CO2 from [5-14C] glutamate and [1-14C] glutamate in mutants from groups II, VII, and the "overlapping" mutant, whereas a significant amount of 14CO2 is released in mutants from group I; (4) a substantial rate of release of 14CO2 from [U-14C] asparate; (5) in isolated mitochondria, succinate and alpha-glycerol phosphate stimulate O2 consumption whereas substrates which generate NADH, such as malate, do not; and (6) there is little or no rotenone-sensitive NADH oxidase activity in isolated mitochondria.     

Respiration-deficient Chinese hamster cell mutants: genetic characterization.

We present here genetic experiments with a series of Chinese hamster cell mutants defective in oxidative energy metabolism. The mutations were all shown to be recessive in intraspecies hybrids. Thirty-five mutants were sorted into eight complementation groups, but one of these mutants failed to complement representatives of two distinct complementation groups. The possibility was raised that this is a cell carrying two mutations or a deletion. Because of the greatly different frequencies with which such mutants could be isolated from two different Chinese hamster cell lines, CCL16 (DON) and V79, the stability of representatives from each cell line was examined, and it was found that revertants could be obtained after treatment with mutagens, while spontaneous revertants appeared at unmeasurable or extremely low frequencies, with one exception. The mutant with a very noticeable frequency of spontaneous reversion was defective in mitochondrial protein synthesis, and the question arose whether the mutation was on the mitochondrial genome. A detailed fluctuation analysis of reversion rate and comparison with rates for other mutations was consistent with a nuclear mutation. This conclusion was supported by experiments involving fusions with cytoplasts.     

Extracellular nitric oxide signaling in the hamster biological clock

Nocturnal light pulses induce phase shifts in circadian rhythms and activate cFos expression in the suprachiasmatic nuclei (SCN). We have studied the role of nitric oxide (NO) in the intercellular communication within the dorsal and ventral portions of the SCN in Syrian hamsters. Administration of the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide blocked photic phase advances in a dose-dependent manner and inhibited light-induced cFos-ir, without affecting light-induced circadian phase delays. These results suggest that NO may act as an intercellular messenger in the SCN, mediating light-induced phase advances.     

Biological clock mutants and parameter changes in mathematical models

A number of recent experiments have revealed the existence of mutants with different free run periods in their circadian rhythms. Parameter variations in mathematical models can be used to simulate such changes. In addition, phase response curves (PRC) are derived and the effect of parameter variation in their shape is studied. It is shown that changes in global parameters can also distort their shape. Therefore one cannot conclude that genetic experiments provide evidence in favor of “chronon” models since “kinetic” models can also simulate their outcome.     

Neuropeptide Y phase advances the in vitro hamster circadian clock during the subjective day with no effect on phase during the subjective night

The mammalian daily (circadian) clock is located in the suprachiasmatic nuclei of the hypothalamus. Clock function can be detected by the measurement of the circadian change in spontaneous firing rate of suprachiasmatic nuclei cells in a brain slice preparation in vitro. We investigated the effects of neuropeptide Y on this rhythm of firing rate in hamster suprachiasmatic nuclei neurons. Slices were prepared using standard techniques. On the 1st day in vitro, neuropeptide Y (200 ng/200 nL; 47 pmol) was applied as a microdrop to the suprachiasmatic nuclei region at various times. Spontaneous single-unit firing was measured for 6-12 h on the 2nd day in vitro. Peak firing rate in treated slices was compared with that of untreated control slices to measure phase shifts induced by the peptide. Neuropeptide Y induced phase advances of circa-3h when applied during the subjective day (ZT 2-10) but did not significantly alter phase when applied during the subjective night. The phase shifts to neuropeptide Y in the hamster tissue in vitro are similar in phase dependency and magnitude to shifts measured in vivo.     

Pesticide induced ouabain resistant mutants in Chinese hamster V79 cells.

The effect of 3 insecticides (chlordane, dieldrin and carbaryl) and one herbicide (2,4-D-fluid) was studied on the induction of ouabain-resistant mutants in Chinese hamster V79 cells at concentrations approaching the Environmental Protection Agency (EPA) tolerance limits. The kinetics and dose range for cytotoxicity were determined by colony formation assay. Results showed that these compounds enhanced the number of ouabain-resistant mutants and acted as weak mutagens.     

Chinese hamster ovary cell mutants defective in the internalization of ricin.

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.     

Myristic acid utilization in Chinese hamster ovary cells and peroxisome-deficient mutants.

Chinese hamster ovary (CHO) cells convert [9,10-3H]myristic acid ([3H]14:0) to several lipid-soluble, radioactive metabolites that are released into the medium. The main products are lauric (12:0) and decanoic (10:0) acids. Some of the 12:0 formed also is retained in cell lipids. Similar metabolites are not synthesized from palmitic (16:0), oleic (18:1), or arachidonic (20:4) acids, and the addition of these fatty acids does not reduce the conversion of [3H]14:0 to 12:0. Two peroxisome-deficient CHO cell lines do not convert [3H] 14:0 to any polar metabolites, but, they elongate, desaturate, and incorporate [3H]14:0 into intracellular lipids and proteins normally. While BC3H1 muscle cells convert some [3H]14:0 to 12:0, they also produce at least nine lipid-soluble polar products from [3H]12:0. These findings suggest that a previously unrecognized function of myristic acid is to serve as a substrate for the synthesis of 12:0, which can be either secreted into the medium or converted to other oxidized metabolites. The absence of this peroxisomal oxidation pathway, however, does not interfere with other aspects of myristic acid metabolism, including protein myristoylation.     

Histone deletion mutants challenge the molecular clock hypothesis

A basic tenet of the molecular clock hypothesis is that the rate of sequence drift for a protein depends on the number of amino acid residues that are critical for its function. However, recent experiments have determined that, although core histone sequences are highly conserved among eukaryotes, large regions of the proteins are dispensable for growth in yeast.     

Altered cell surface glycoproteins in phytohemagglutinin-resistant mutants of Chinese hamster ovary cells.

Purified membranes from surface-labelled phytohemagglutinin-resistant (Pha(R) and wild-type chinese hamster ovary cells have been analysed by sodium dodecyl sulphate gel electrophoresis. Gel patterns were compared for cells labelled via galactose oxidase and B-3H4 or lactoperoxidase and radioactive iodide. The results suggest that Pha-R cells are altered in the carbohydrate portion of a number of their membrane glycoproteins.     

Chinese hamster ovary cell mutants defective in myo-inositol transport

By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.