Intrapreoptic microinjection of GHRH or its antagonist alters sleep in rats.
Previous reports indicate that growth hormone-releasing hormone (GHRH) is involved in sleep regulation. The site of action mediating the nonrapid eye movement sleep (NREMS)-promoting effects of GHRH is not known, but it is independent from the pituitary. GHRH (0.001, 0. 01, and 0.1 nmol/kg) or a competitive antagonist of GHRH (0.003, 0.3, and 14 nmol/kg) was microinjected into the preoptic area, and the sleep-wake activity was recorded for 23 hr after injection in rats. GHRH elicited dose-dependent increases in the duration and in the intensity of NREMS compared with that in control records after intrapreoptic injection of physiological saline. The antagonist decreased the duration and intensity of NREMS and prolonged sleep latency. Consistent alterations in rapid eye movement sleep (REMS) and in brain temperature were not found. The GHRH antagonist also attenuated the enhancements in NREMS elicited by 3 hr of sleep deprivation. Histological verification of the injection sites showed that the majority of the effective injections were in the preoptic area and the diagonal band of Broca. The results indicate that the preoptic area mediates the sleep-promoting activity of GHRH. (+info)
Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin.
In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect. (+info)
Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.
Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK-/- rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK-/- mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK-/- mice raised in constant darkness. One day of constant light caused the rods in the RK-/- mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness. (+info)
Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression.
Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression. (+info)
Light-induced oxidation-reduction reactions of cytochromes in the green sulfur photosynthetic bacterium Prosthecochloris aesturarii.
The light-induced oxidation-reduction reactions of cytochromes in intact cells, starved cells, and chlorobium vesicle fractions of the green sulfur photosynthetic bacterium Prosthecochloris aesturarii were studied under anaerobic conditions. On the basis of both kinetic and spectral properties, at least three cytochrome species were found to be involved in the light-induced oxidation-reduction reactions of intact cells. These cytochromes were designated according to the positions of alpha-band maxima as C555 (rapid and slow components) and C552 (intermediate). By comparing the light-minus-dark difference spectra with the reduced-minus-oxidized difference spectra of purified cytochromes of this organism, rapid component C555 and intermediate component C552 are suggested to correspond to the purified cytochromes c-555(550) and c-551.5, respectively. Although the identity of the slow-phase component is uncertain, one possibility is that the slow phase is due to the bound form of c-555(550). In substrate-depleted (starved) cells, only one cytochrome species, C555 remained in the reduced state in the dark and oxidized upon actinic illumination. This corresponds to the rapid C555 component in intact cells. In the case of chlorobium vesicle fractions, one cytochrome species having an alpha-band maximum at 554 nm was oxidized by actinic light. The effects of several inhibitors on the absorbance changes of intact cells were studied. Antimycin A decreased the rate of the dark reduction of rapid C555 component. The complex effects of CCCP (carbonyl cyanide m-chlorophenylhydrazone) on the oxidation-reduction reactions of cytochromes were interpreted as the results of inhibition of the electron donation to oxidized C552 and C555 (slow), and a shift of the dark steady-state redox levels of cytochromes. Based on these findings, it is suggested that the rapid C555 component is located in a cyclic electron transfer pathway. The other two cytochromes, C552 and C555 (slow), may be located in non-cyclic electron transfer pathways and receive electrons from exogenous substrates such as sodium sulfide. A tentative scheme for the electron transfer system in Prosthecochloris aestuarii is presented and its nature is discussed. (+info)
Modulation of Hydra attenuata rhythmic activity: phase response curve.
We investigated the effect of photic stimulation on the frequency of Hydra attenuata column contractions. We used positive or negative abrupt light transitions, single or repetitive light or darkness pulses, and alternation of light and darkness periods. The main results are: (a) The frequency of the contraction pulse trains (CPTs) varies transiently in response to an abrupt variation of the light intensity. (b) CPTs in progress can be inhibited by different types of photic stimuli. (c) The response time to a single photic stimulus varies during the inter-CPT interval and depends also on the polarity of the stimulus. (d) The CPTs are entrainable with repetitive light stimulation of various frequencies. (e) Long-lasting variations of the frequency of CPTs occur after the end of a repetitive light stimulation. We suggest that the mechanism responsible for the rhythym of column contractions is quite similar to that on which other biological rhythmic phenomena are based. (+info)
The relation between intercellular coupling and electrical noise in turtle photoreceptors.
1. Intracellular recordings from cones and rods in the retina of the turtle, Pseudemys scripta elegans, revealed that in darkness the cell voltage fluctuated spontaneously about its mean level. The fluctuations were reduced during bright steady illmination of the cell often to a level close to that obtained with the electrode outside the cell where the noise did not change significantly during illumination. 2. The magnitude of the intrinsic dark noise (voltage variance in darkness minus voltage variance in strong light) varied widely from cell to cell. In the noisiest cones it was about 0-4 mV2 while in quiet cones it was often as low as 0-01 mV2. The noise appeared radom and could be fitted by a Gaussian probability density function. 3. The spread of voltage in the network of coupled photoreceptors was estimated by measuring the spatial profile of the response to a brief flash of constant intensity moved across the retina. For a light stimulus in the form of a long narrow slit, the peak flash response usually decayed exponentially with displacement from the centred position. 4. For maximum responses less than about 5 mV in cones, the length constant of exponential decay, lambda, varied from less than 10 mum to greater than 35 mum, and the values obtained in opposite directions were often unequal. Background illumination did not significantly change lambda. In cells with extremely narrow spatial profiles, an exponential fit to the decay could not be made reliably. 5. Occasionally the spatial profiles had definite secondary peaks. In the most pronounced examples in a red-sensitive cone and in a rod the maxima were separated by about 20 and 50 mum respectively; for each, one peak was approximately as sharp as the optical stimulator while the second was broader. 6. Cones with short length constants displayed high dark noise while cones with long length constants were relatively quiet. 7. Three models of electrical coupling between cells were investigated: one based on a distributed network, one on a discrete square grid arrangement, and one on a discrete hexagonal array. Each model predicts a strong dependence of both noise and input resistance on length constant, and for tightly coupled cells each predicts that voltage variance is proportional to lambda-2. 8. The measured relationship between voltage variance and lambda in a large sample of cones was well described by both discrete models when the average cell spacing was taken to be approximately 15 mum. 9... (+info)
The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.
Post-translational attachment of isoprenyl groups to conserved cysteine residues at the C-terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C-terminal basic domain and a CTIL CaaX-box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild-type CaM53 or a non-prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non-prenylated mutant protein localizes to the nucleus, indicating a dual role for the C-terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities. (+info)