Biosynthesis of a microbody matrix enzyme in greening cotyledons

Earlier work on microbody biosynthesis has shown that glyoxysomal and liver peroxisomal proteins synthesized in the cytosol are made as precursors which are then transferred into the organelles and processed. Here, it is demonstrated that the unprecessed precursor detected in the cytosol after protein synthesis in vivo for an enzyme at the transition stage between glyoxysomes and leaf peroxisomes is indistinguishable from the product of translation in vitro. It is assumed that the transfer of extraorganellarly made precursor across the glyoxysomal membranes is followed by processing of the precursor and oligomerization to the tetrameric or 16-meric form of the enzyme. Oligomerization was, however, also observed in a portion of the cytosolic form.     

A rapid circadian rhythm of carbon-dioxide metabolism in Bryophyllum fedtschenkoi

Leaves of Bryophyllum fedtschenkoi Hamet et Perrier maintained in a stream of normal air and at 15° C exhibit a circadian rhythm of CO₂ uptake in continuous light but not in continuous darkness. The rhythm is unusual in that it persists for at least 10 d, and has a short period of approximately 18 h. The mechanism by which this rhythm is generated is discussed.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals. This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.     

The action of exogenous gibberellic acid on protein and mRNA in germinating castor bean seeds

Gibberellic acid (GA₃) stimulates water uptake in castor beans and increases the activity of certain enzymes associated with lipid mobilisation.The effect of the GA₃ on the enzymes is possibly due to a general effect of the growth substance on protein synthesis. Gibberellic acid advanced the appearance of rRNA and poly (A⁺)RNA in castor bean endosperms without specifically stimulating the synthesis of particular mRNA species. Thus these increased levels of mRNA and rRNA may act synergistically to affect the rate of a predetermined pattern of protein synthesis.Abbreviations SDS sodium dodecyl sulphate - GA₃ gibberellic acid - PAGE polyacrylamide gel electrophoresis     

Establishing the presence of a body temperature rhythm in chimpanzees (Pan troglodytes) using a tympanic membrane thermometer

Seven chimpanzees (Pan troglodytes) were trained to present their ears so that a tympanic nembrane thermometer could be inserted. Temperatures were collected from both ears of each subject every 3 hours for 72 consecutive hours. The presence of a body temperature rhythm, well documented in other mammals, was established. Each ear demonstrated its own rhythm, but the rhythms in both ears generally mirrored each other. Similarities in the temperature rhythms of cagemates were found. These data are the first evidence of a body temperature rhythm in chimpanzees, and they represent a non-invasive method of measuring the 24-hr rhythms in both human and non-human primates.     

Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1^-1 [-³²P]ATP whereas incubation with 50 mol·1^-1 [-³²P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1^-1 ATP, and around 40 mol·1^-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-³²P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1^-1). The ADP-dependent appearance of ³²P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of ³²P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1^-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.Abbreviations LHCP ligh-harvesting chlorophyll-a/b-binding protein - S_0.5 concentration giving half-maximal phosphorylation - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Temperature-induced phase shifting of circadian rhythms in cotton seedlings as related to variations in chilling resistance

The relationship between the degree of chilling resistance and phase shifting caused by low-temperature pulses was examined in two circadian rhythms in cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 1212 h at 33° C. The seedlings showed a circadian rhythm of chilling resistance and of cotyledon movement. A pulse of 19° C for 12 h during the chilling-sensitive phase (light period) caused a phase delay of 6 h, while a similar temperature pulse during the chilling-resistant phase (dark period) did not cause any phase shift. Exposure to 19° C, 85% RH (relative humidity) for 12 h during the dark period induced chilling resistance in the following otherwise chilling-sensitive light period. In this light period a 12-h 19° C pulse did not cause a phase shift of chilling resistance. Pulses of low temperatures (5–19° C) were more effective in causing phase delays in the rhythm of cotyledon movement when given during the chilling-sensitive phase than when given during the chilling-resistant phase. A 12-h pulse of 5° C, 100% RH during the light period caused a phase delay of cotyledon movement of 12 h. However, when that pulse had been preceded by a chill-acclimating exposure to 19° C, 85% RH for 12 h during the dark period the phase delay was shortened to 6 h. The correlation between higher degree of chilling resistance and the prevention or shortening of the phase delay caused by low temperatures indicates that the mechanism that increases chilling resistance directly or indirectly confers greater ability for prevention of phase shifting by low temperatures in circadian rhythms.Abbreviations CT circadian time - LDC light-dark cycle of 24 h - RH relative humidity     

Endogenous oscillator controls surface density of mating receptors in Chlamydomonas eugametos

Summary We describe a circadian rhythm in the surface density of receptors that play a dominant role in the mating process of the unicellular green alga Chlamydomonas eugametos.These receptors — called agglutinins — are large glycoproteins extrinsically bound to the membrane of gamete flagella. We found circadian fluctuations in their density. Since inhibition of protein synthesis affected the agglutinin density without a lag period at any time,we conclude that the density was dependent on de novo synthesis and that the fluctuations in density are caused by circadian oscillations in the rate of agglutinin synthesis. This phenomenon evidently underlies the pronounced endogenous rhythm in mating competence that we described previously (Demets et al. 1987). Finally, we speculate on the nature of the time keeping mechanism that is generating these rhythmic events.     

Body temperature and behaviour of golden hamsters (Mesocricetus auratus) and ground squirrels (Spermophilus tridecemlineatus) in a thermal gradient

Golden hamsters and thirteen-lined ground squirrels were maintained individually in a thermal gradient (14°C to 33°C) for several weeks under a 14L: 10D light-dark cycle. Animals of both species showed robust daily rhythms of body temperature and locomotor activity with acrophases consistent with the habits of the species (diurnal acrophases in the diurnal squirrels and nocturnal acrophases in the nocturnal hamsters). Hamsters showed a robust daily rhythm of temperature selection 180° out of phase with the rhythms of body temperature and locomotor activity. Squirrels did not show a daily rhythm of temperature selection. These results raise the hypothesis that a daily rhythm of temperature selection is exhibited by nocturnal but not by diurnal endotherms.     

Circadian control of heat tolerance in stationary phase cultures of Schizosaccharomyces pombe

The capacity of stationary phase cultures of Schizosaccharomyces pombe to survive a heat treatment at 55°C is controlled by a circadian rhythm. In a synchronizing light-dark-cycle this rhythm shows a stable phase relationship to the onset of light. In continuous darkness it persists for several cycles without marked damping. The free-running period of about 27 h at 30°C is only slightly longer at 20°C, hence temperature-compensated. These results indicate that S. pombe is a suitable experimental organism for further research into both heat tolerance and circadian rhythms.     

In vitro synthesis of the cyanelle proteins of Cyanophora paradoxa by isolated cyanelles and cyanelle RNA

Cyanelles isolated from the alga Cyanophora paradoxa Korschikoff synthesized cyanelle proteins in vitro. This synthesis was stimulated by light and totally inhibited by chloramphenicol. Cycloheximide had only a small inhibitory effect. Electrophoretic separation of the labelled soluble cyanelle proteins yielded at least 20 discrete polypeptides. The RNA isolated from the cyanelles and the whole cells was successfully translated in a rabbit reticulocyte-lysate system.Abbreviations poly(A)^-RNA, poly(A)⁺RNA nonadenylated, polyadenylated RNA; - SDS sodium dodecyl sulfate