The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat-shocked Drosophila cells. However, at 36-37 degrees C, the optimum temperature for induction of the 70-kDa heat-shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three-hour period. Incubation of heat-shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat-shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat-shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat-shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat-shock response in this organism, which makes it unlikely that the mechanism of this acute shut-off is intimately connected with the mechanism of induction of heat-shock mRNAs.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Induction of four heat-shock proteins and their mRNAs in rat after whole-body hyperthermia

When the body temperature of rats was brought to 42 degrees C, four heat-shock proteins, with molecular weights of 70,000, 71,000, 85,000, and 100,000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), were induced in various tissues of the rats. The hsp 70 was strongly induced by hyperthermia, and its accumulation was detected by Coomassie blue staining. The hsp 71 was abundant in various tissues of rats that were not heat-shocked. Analysis of translation products of liver mRNAs from heat-shocked rats also showed increased synthesis of the four heat-shock proteins, indicating that these hsp-mRNAs were induced after hyperthermia. Induction of the hsp-mRNAs was transient after hyperthermia. The four heat-shock proteins produced in various tissues after hyperthermia may be involved in homeostatic control in the heat-shock response of the rat.     

Varying patterns of protein synthesis in Drosophila during heat shock: Implications for regulation

The heat-shock response of Drosophila involves the vigorous induction of a small number of new messenger RNAs and proteins as well as the repression of most preexisting RNA and protein synthesis. The experiments presented here characterize the kinetics of messenger RNA and protein synthesis at different temperatures and the patterns of induction under a variety of culture conditions. In addition to providing practical information for further studies of the heat-shock response, the data provide some valuable insights into the nature of the response. In particular, the patterns of induction and repression are not simple functions of the degree of temperature elevation, but vary strikingly in different media and depend strongly upon the speed of the temperature increase. Several heat-shock proteins are shown to have very individual induction characteristics with respect to the temperatures at which they are maximally induced, the range of temperatures over which they are synthesized, and the kinetics of their induction. Thus, although this system has often been viewed as a simple, coordinate induction, it now appears that the various heat-shock genes can be, to a rather considerable extent, regulated independently of one another. The evidence further suggests that the patterns of protein synthesis in heat-shocked cells are regulated by mechanisms which act at several different levels of gene expression.     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

Synthesis of heat shock proteins in Drosophila melanogaster embryos

The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation.     

A circadian rhythm in neural activity can be recorded from the central nervous system of the cockroach

Summary Evidence presented in this paper indicates that a robust circadian rhythm in the frequency of neural activity can be recorded from the central nervous system of intact cockroaches, Leucophaea maderae. This rhythmicity was abolished by optic lobe removal. Spontaneous neural activity was then used as an assay to demonstrate that the optic lobe is able to generate circadian oscillations in vitro. These results provide direct evidence that the cockroach optic lobe is a self-sustained circadian oscillator capable of generating daily rhythms in the absence of neural or hormonal communications with the rest of the organism.Abbreviations CNS central nervous system - DD constant dark - LD light/dark cycle - SCN suprachiasmatic nucleus - ZT Zeitgeber time     

Feeding rhythms in constant light and constant darkness: the role of the eyes and the effect of melatonin infusion

Exposure to constant light abolishes circadian behavioral rhythms of locomotion and feeding as well as circulating melatonin rhythms in pigeons (Columba livia). To determine if feeding rhythmicity could be maintained in pigeons exposed to constant light, periodic infusions (10h/day) of melatonin were administered to pinealectomized and bilaterally retinectomized/pinealectomized pigeons under conditions of both constant darkness and constant light. The infusions were sufficient to entrain rhythmicity in pinealectomized pigeons in constant darkness and to restore and maintain rhythmicity in bilaterally retinectomized/pinealectomized pigeons in constant darkness. On subsequent exposure to constant light, rhythmicity remained phase locked to the melatonin infusions in bilaterally retinectomized/pinealectomized pigeons but was abolished in sighted pinealectomized birds. These results suggest that while endogenous melatonin rhythms are both necessary and sufficient to maintain behavioral rhythms in DD, their effect can be overridden by constant light but only if perceived by the eyes. Thus, constant light may abolish behavioral rhythmicity in intact pigeons (and perhaps in other species) by a mechanism other than suppression of endogenous melatonin rhythmicity. Such a mechanism might involve direct stimulation of locomotor or feeding activity by retinally perceived (but not by extra-retinally perceived) light, or alternatively by suppression of a hypothalamic oscillator that receives its major light input from the retinae.Abbreviations PX pinealectomized - EX bilaterally enucleated - LD light:dark cycle - LL constant light - DD constant darkness - DD_b constant darkness before exposure to constant light - DD_a constant darkness after exposure to constant light     

Overexpression of White Collar-1 (WC-1) activates circadian clock-associated genes,but is not sufficient to induce most light-regulated gene expression in Neurospora crassa

Many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. The White Collar-1 (WC-1) protein is required for all known blue-light responses in Neurospora crassa. In response to light, WC-1 levels increase, and the protein is transiently phosphorylated. To test the hypothesis that the increase in WC-1 levels after light treatment is sufficient to activate light-regulated gene expression, we used microarrays to identify genes that respond to light treatment. We then overexpressed WC-1 in dark-grown tissue and used the microarrays to identify genes regulated by an increase in WC-1 levels. We found that 3% of the genes were responsive to light, whereas 7% of the genes were responsive to WC-1 overexpression in the dark. However, only four out of 22 light-induced genes were also induced by WC-1 overexpression, demonstrating that changes in the levels of WC-1 are not sufficient to activate all light-responsive genes. The WC proteins are also required for circadian rhythms in dark-grown cultures and for light entrainment of the circadian clock, and WC-1 protein levels show a circadian rhythm in the dark. We found that representative samples of the mRNAs induced by over-expression of WC-1 show circadian fluctuations in their levels. These data suggest that WC-1 can mediate both light and circadian responses, with an increase in WC-1 levels affecting circadian clock-responsive gene regulation and other features of WC-1, possibly its phosphorylation, affecting light-responsive gene regulation.     

Reliability of the Circadian Rhythm of Water and Macronutrient-Rich Diets Intake in Dietary Choice

Rats with ad libitum water and the ability to self-select among three macronutrient-rich diets—carbohydrate (CHO), protein (PRO), and lipid (LIP)—show a circadian rhythmicity in their ingestion. The aim of the present study was to determine whether this circadian rhythmicity is reliable from day to day. Eight rats were offered ad libitum water and a choice of three isoenergetic diet rations providing carbohydrate, protein, and lipid. Water and food intake was recorded every 3 h for 7 days. The reliability of the circadian rhythm of water and food intake was assessed by the Intraclass Correlation Coefficient (ICC) and the test-retest reliability using the Pearson's Correlation Coefficient (r). The results showed that the circadian rhythm of water, CHO, and PRO intake are strongly reliable. However, the circadian rhythm of LIP intake is less reproducible. Among the three reliable parameters—water, CHO, and PRO, the circadian rhythm of water intake was the most reproducible over 7 days. This suggests that water intake may be used as a marker of circadian rhythmicity in ingestive behavior.     

Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia

Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89-kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

Circadian Control of the Accumulation of mRNAs for Light- and Heat-Inducible Chloroplast Proteins in Pea (Pisum sativum L.)

The levels of the mRNAs for light-inducible, nuclear-coded chloroplast proteins vary rhythmically in pea (Pisum sativum L.) plants either grown in a dark-light cycle or under constant light conditions. This has been observed for the early light-inducible protein, the light-harvesting chlorophyll a/b protein, and the small subunit of the ribulose-1,5-bisphosphate carboxylase. The mRNA levels are high in the morning, exhibit a minimum in the first half of the night, and increase again during the second half of the night. The amplitude of fluctuation is between 5- and 10-fold. A similar change in the mRNA abundance was found for four nuclear encoded heat-shock proteins of 18, 24, 26, and 30 kilodaltons. The ability of plants to transcribe heat-shock genes upon heat-shock for 2 hours varies through the day. The maxima for induction are found in the second half of the night and the morning. The minima are reached during the afternoon. The degree of fluctuation is between 3- and 5-fold. The levels of mRNAs for cytosolic as well as for plastid heat-shock proteins oscillate in parallel.     

Chenopodium rubrum as a model plant for testing the flowering effects of PGRs

In short-day plantChenopodium rubrum the photoperiodic requirement for flowering increases from one short day at the age of four days to two or three short days at the age of five to seven days. The photoperiodic requirement decreases again to one short day between the 10th and 12th day of cultivation. This feature, together with endogenous circadian rhythmicity of flowering, enabled us to test the effects of PGRs under different morphogenetic patterns. Representative PGR effects on flowering are quoted.     

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich cells involves reduction of eukaryotic initiation factor 4F activity

Almost all living organisms studied respond to elevated temperature with a marked inhibition of overall protein synthesis but increased synthesis of a specific set of proteins, the so-called heat-shock proteins. We have prepared a cell-free protein synthesizing system (lysate) from heat-shocked Ehrlich ascites tumor cells that reflects the inhibition of protein synthesis in intact cells at elevated temperatures. We have isolated and partially purified a stimulator of the heat-shocked cell lysate from Ehrlich cells. Through four purification steps, the stimulator is chromatographically identical to eukaryotic initiation factor 4F (eIF-4F), an initiation factor which specifically binds mRNA cap structure. Therefore, we have tested the effects of highly purified reticulocyte eIF-4F on the heat-shocked cell lysate. Protein synthesis is strongly stimulated by addition of highly purified eIF-4F. Synthesis in the heat-shocked lysate is more inhibited at high (70 mM) KCl concentrations, than at lower concentrations, and stimulation by eIF-4F is correspondingly greater at higher KCl concentrations, so that the rate of protein synthesis is returned to control (non-heat-shocked lysate) levels at all KCl concentrations. Furthermore, at 70 mM KCl, in heat-shocked lysates, synthesis of the 68-kDa heat-shock protein is much less inhibited than synthesis of the bulk of non-heat-shock proteins, and eIF-4F stimulates synthesis of 68-kDa protein to a much lesser extent than non-heat-shock proteins. Thus, addition of purified eIF-4F reverses the effects of elevated temperatures on Ehrlich cells that are reflected in lysates. Therefore, we propose that the inhibition of translation in heat-shocked Ehrlich cells is the result of inactivation of eIF-4F function.     

Gradual reappearance of post-hibernation circadian rhythmicity correlates with numbers of vasopressin-containing neurons in the suprachiasmatic nuclei of European ground squirrels

European ground squirrels (Spermophilus citellus) in outside enclosures show suppressed circadian rhythmicity in body temperature patterns during the first days of euthermia after hibernation. This may reflect either gradual reappearance of circadian rhythmicity following suppressed functioning of the circadian system during hibernation, or it may reflect transient days during re-entrainment of the circadian system which, during hibernation, has drifted out of phase with the environmental light-dark cycle. Here we report that animals kept under continuous dim light conditions also showed absence of circadian rhythmicity in activity and body temperature in the first 5-15 days after hibernation. After post-hibernation arrhythmicity, spontaneous circadian rhythms re-appeared gradually and increased daily body temperature range. Numbers of arginine-vasopressin immunoreactive neurons in the suprachiasmatic nuclei correlated positively with individual circadian rhythmicity and increased gradually over time after hibernation. Furthermore, circadian rhythmicity was enhanced rather than suppressed after exposure to a light-dark cycle but not after a single 1-h light pulse (1,700 lux). The results support the view that the functioning of the circadian system in the European ground squirrel is suppressed during hibernation at low temperatures and that it requires several days of euthermia to resume its summer function.     

Insect circadian rhythms and photoperiodism

Two clock-controlled processes, overt circadian rhythmicity and the photoperiodic induction of diapause, are described in the blow fly,Calliphora vicina and the fruit fly,Drosophila melanogaster. Circadian locomotor rhythms of the adult flies reflect endogenous, self-sustained oscillations with a temperature compensated period. The free-running rhythms become synchronised (entrained) to daily light:dark cycles, but become arrhythmic in constant light above a certain intensity. Some flies show fragmented rhythms (internal desynchronisation) suggesting that overt rhythmicity is the product of a multioscillator (multicellular) system. Photoperiodic induction of larval diapause inC. vicina and of ovarian diapause inD. melanogaster is also based on the circadian system but seems, to involve a separate mechanism at both the molecular and neuronal levels. For both processes in both species, the compound eyes and ocelli are neither essential nor necessary for photic entrainment, and the circadian clock mechanism is not within the optic lobes. The central brain is the most likely site for both rhythm generation and extra-optic photoreception. InD. melanogaster, a group of lateral brain neurons has been identified as important circadian pacemaker cells, which are possibly also photo-sensitive. Similar lateral brain neurons, staining for arrestin, a protein in the phototransduction ‘cascade’ and a selective marker for photoreceptors in both vertebrates and invertebrates, have been identified inC. vicina. Much less is known about the cellular substrate of the photoperiodic mechanism, but this may involve thepars intercerebralis region of the mid-brain.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.