Circadian clock mutants in Arabidopsis identified by luciferase imaging.

The cycling bioluminescence of Arabidopsis plants carrying a firefly luciferase fusion construct was used to identify mutant individuals with aberrant cycling patterns. Both long- and short-period mutants were recovered. A semidominant short-period mutation, timing of CAB expression (toc1), was mapped to chromosome 5. The toc1 mutation shortens the period of two distinct circadian rhythms, the expression of chlorophyll a/b-binding protein (CAB) genes and the movements of primary leaves, although toc1 mutants do not show extensive pleiotropy for other phenotypes.

Conditional circadian dysfunction of the Arabidopsis early-flowering 3 mutant.

Photoperiodic responses, such as the daylength-dependent control of reproductive development, are associated with a circadian biological clock. The photoperiod-insensitive early-flowering 3 (elf3) mutant of Arabidopsis thaliana lacks rhythmicity in two distinct circadian-regulated processes. This defect was apparent only when plants were assayed under constant light conditions. elf3 mutants retain rhythmicity in constant dark and anticipate light/dark transitions under most light/dark regimes. The conditional arrhythmic phenotype suggests that the circadian pacemaker is intact in darkness in elf3 mutant plants, but the transduction of light signals to the circadian clock is impaired.

Multiple DNA-Protein Complexes at a Circadian-Regulated Promoter Element.

Higher plant CAB genes encode chlorophyll a/b binding proteins that are part of light-harvesting complexes in chloroplasts. Transcription of the Arabidopsis CAB2 (lhcb1*1) gene is under the control of a circadian oscillator and exhibits high amplitude diurnal oscillations that persist within a period close to 24 hr in the absence of environmental time cues. Initial deletion studies in transgenic tobacco have demonstrated that the region between -111 and -38 of the CAB2 promoter sequence confers circadian regulation to a luciferase (luc) reporter gene. We dissected this element further and characterized five DNA binding complexes from Arabidopsis whole-cell extracts that bind within this region of the promoter and may be components of the signal transduction pathway for the control of transcription by the circadian clock. The in vivo analysis of cab2::luc fusion constructs in transgenic Arabidopsis demonstrated that a circadian-regulated element lies within a 36-bp sequence that overlaps a conserved CCAAT box and contains binding sites for three putative transcription factors.

Day-length perception and the photoperiodic regulation of flowering in Arabidopsis.

The flowering of Arabidopsis plants is accelerated by long-day photoperiods, and recent genetic studies have identified elements of the photoperiodic timing mechanism. These elements comprise genes that regulate the function of the circadian clock, photoreceptors, and downstream components of light signaling pathways. These results provide evidence for the role of the circadian clock in photoperiodic time measurement and suggest that photoperiod perception may follow Pittendrigh's external coincidence model. T-cycle experiments indicated that changes in the timing of circadian rhythms, relative to dawn and dusk, correlated with altered flowering time. Thus, the perception of photoperiod maybe mediated by adjustments in the phase of the circadian cycle that arise upon re-entrainment to a different light-dark cycle. The nature of the rhythm underlying the floral response is not known, but candidate molecules have been identified.

In vitro O2-induced depression of T and B lymphocyte activation is reversed by diethyldithiocarbamate (DDC) treatment.

In this study, we tried to establish a relationship between the immunopotentiating effects and the antioxidant activity of the immunostimulating compound, diethyldithiocarbamate (DDC). We studied the effects of DDC treatment on enriched T and B murine spleen lymphocytes in an in vivo-ex vivo model of O2-induced immune depression. Female C57B1/6 mice were injected subcutaneously with a single dose of DDC (125 mg.kg-1). Eight days after DDC injection, we evaluated, in vitro, the concanavalin A response of the T cell fraction and the LPS response of the B cell fraction, under standard (air--5% CO2) and hyperoxic (60% O2--5% CO2) culture conditions. The results show that after a lag period, DDC is able to enhance the mitogenic response of T and B murine lymphocytes under standard culture conditions to restore the ConA response and to partially restore the LPS response under hyperoxic conditions. The results of this study suggest that the immunostimulatory effects of DDC could be related to the antioxidant activity of this compound on the lymphoid cellular metabolism. This activity apparently affects both T and B lymphocytes.

The role of ions and second messengers in circadian clock function.

The fact that single cells can exhibit circadian rhythmicity simultaneously in quite different processes, such as those of photosynthesis, bioluminescence, and cell division, suggests that membrane-bound compartmentalization is important for temporal organization. Since these rhythms, as well as others, are known to be affected by changes in the ionic environment and are probably membrane-bound systems, it is not surprising that transmembrane ion transport or flux has been proposed to be a key feature of the underlying circadian oscillator(s). Likewise, signal transduction along the entrainment pathway leading to the clock, among the elements, or "gears," of the timing loop itself, and within the output pathway between the oscillator and its "hands" likely is mediated by ions and second messengers. In this overview, we examine the theoretical and experimental evidence supporting the possible roles of intracellular free calcium and cyclic AMP in these capacities, particularly in view of the fact that oscillations in the concentrations of both species have been proposed to form the basis of pacemaker activity and other biological rhythms.

Circadian variations in the affinities of NAD kinase and NADP phosphatase for their substrates, NAD+ and NADP+, in dividing and nondividing cells of the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z).

We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP+ and NAD+, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CT), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2(+)-calmodulin) or for its substrate, NAD+. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.

A 20-year prospective follow-up of childhood hiatal hernia.

A long-term prospective follow-up of 113 children with vomiting due to a small hiatal hernia is described. When reviewed by the same clinical and radiological observers 20 or more years later, over 90% of unoperated non-stricture patients were asymptomatic whereas only 44% of the stricture and/or surgically treated group were without symptoms. Half or possibly more of the asymptomatic patients still had a hernia and it is possible that these may suffer a recurrence of symptoms later in adult life. The loculus of thoracic stomach tended to retain the same shape; there was a slightly better prognosis for the locular type of hernia compared with the tubular type. Complicating oesophageal strictures can decrease or disappear without surgery other than dilatation; the results of treatment by radical surgery were disappointing. There is need for an even more prolonged follow-up into later adult life.

Regulation of a NAD+ kinase activity isolated from asynchronous cultures of the achlorophyllous ZC mutant of Euglena gracilis.

NAD+ kinase was isolated by chromatography steps from asynchronous cultures of the achlorophyllous ZC mutant of Euglena gracilis. A non Ca(2+)-calmodulin dependent form whose activity was stimulated by EGTA, was selected for its large quantity and high specific activity. Studies of the kinetic parameters revealed two kinds of NAD+ binding site, depending on NAD+ concentrations, and changes induced by EGTA, Ca2+ and Ca(2+)-calmodulin. The search for effectors, soluble (S) and membrane-bound (P), in Euglena gracilis synchronously grown (in a light-dark regime of 12h:12h), and collected at circadian times (CT)--corresponding to the maximum, CT 17, and to the trough, CT 09, of the circadian rhythm of NAD+ kinase activity--was also undertaken by testing the modulations of the kinetic parameters of the prepared NAD+ kinase. The results suggest: (i) structural changes of NAD+ binding sites depending on NAD+ concentrations; (ii) possible binding of the Mg-ATP-2 (or Ca-ATP-2) on the NAD+ sites, because of their common ADP motif; and (iii) different and specific modulations of the kinetic parameters of the two types of NAD+ binding site by the Ca(2+)-calmodulin complex. In addition, the results indicate, in pelletable fractions isolated at CT 09 and CT 17, the presence of two kinds of effector:(i) the first one, possibly Ca2+, which increases the Vmax's while decreasing the binding of NAD+; (ii) the second one, possibly the Ca(2+)-calmodulin complex, which provokes a complete reverse effect. Each of these two effectors seems to be, alternatively and rhythmically (eight circadian hours apart), partially released from the membranes.

Management of gastro-oesophageal reflux.

Parental reassurance and thickened feeds are the only requirements in the management of infants with reflux when this is the sole detectable gastro-oesophageal abnormality. In view of the strong propensity for spontaneous clinical resolution and the excellent results achieved by conservative management, infants with reflux due to a partial thoracic stomach (hiatal hernia) uncomplicated by a stricture should be treated in the first instance by postural therapy, with or without thickened feeds and supplements of antacids, domperidone, and cimetidine. Those showing no response after an adequate period of conservative treatment should have an antireflux operation. The Belsey MK IV type of fundoplication is preferred. Only an exceptional patient will require to be treated surgically before 12 months of age. The same surgical antireflux procedure, combined with oesophageal dilatations as necessary, is the treatment of choice for patients with a partial thoracic stomach complicated by a reflux oesophageal stricture. A similar treatment regimen should be followed for patients with reflux after repair of an oesophageal atresia. Surgical correction is mandatory for all infants with reflux due to a large combined hiatal hernia.

Findings on barium swallow in younger siblings of children with hiatal hernia (partial thoracic stomach).

The results are presented of an investigation by barium swallow of 271 younger siblings of children with hiatal hernia (HH). The incidence of HH among younger siblings with a history of repeated vomiting and regurgitation was 41% and in those without symptoms was 4.3%.

Oscillator control of cell division in Euglena: cyclic AMP oscillations mediate the phasing of the cell division cycle by the circadian clock.

The achlorophyllous ZC strain of Euglena gracilis exhibits a circadian rhythm of cell division in constant darkness (DD). Mitosis occurs during a restricted part of the circadian cycle, corresponding to the dark intervals in a light-dark cycle comprising 12 h of light and 12 h of darkness. We have demonstrated that division-phased cultures also exhibit bimodal, circadian changes of cyclic AMP level. Maximum cyclic AMP levels occurred at the beginning of the light period (CT (circadian time) 00-02), and at the beginning of darkness (CT 12-14). These variations persisted in cultures that had been transferred into DD and appeared to be under the control of the circadian oscillator rather than to be cell division cycle (CDC)-dependent, since they continued in cultures that had reached the stationary phase of growth. In the experiments reported in this paper, we tested for the possible role of this periodic cyclic AMP signal in the generation of cell division rhythmicity by examining the effects of exogenous cyclic AMP signals and of forskolin, which permanently increased the cyclic AMP level, on the cell division rhythm. Perturbations of the cyclic AMP oscillation by exogenous cyclic AMP resulted in the temporary uncoupling of the CDC from the circadian timer. The addition of cyclic AMP during the subjective day resulted in delays (up to 9 h) of the next synchronous division step. In contrast, mitosis was stimulated when cyclic AMP was administered in the middle of the subjective night. Measurement of the DNA content of cells by flow cytometry indicated that cyclic AMP injected at CT 06-08 delayed progression through S phase, and perhaps also through mitosis. When added at CT 18-20, cyclic AMP accelerated the G2/M transition. The circadian oscillator was not perturbed by the addition of exogenous cyclic AMP: the division rhythm soon returned to its original phase. On the other hand, the permanent elevation of cyclic AMP levels in the presence of forskolin induced a rapid loss of cell division rhythmicity. These findings are consistent with the hypothesis that cyclic AMP acts downstream from the oscillator and that the cyclic AMP oscillation is an essential component of the signaling pathway for the control of the CDC by the circadian oscillator. The receptors for cyclic AMP in Euglena have been shown to be two cyclic AMP-dependent kinases (cPKA and cPKB). Pharmacological studies using cyclic AMP analogs suggested that cPKA mediates cyclic AMP effects during the subjective day, and cPKB during the subjective night.(ABSTRACT TRUNCATED AT 400 WORDS)

cAMP-dependent kinases in the algal flagellate Euglena gracilis.

Euglena cells grown in diurnal light-dark cycles exhibit circadian variations of their cAMP content, which we believe to be under the control of an endogenous timer because they persist in constant darkness in the absence of any environmental time cue. We think that these cAMP oscillations may play a role in the regulation of some of the numerous cellular activities that are known to display circadian rhythmicities in this organism. The role of cAMP in algal cells is still controversial, however, since the nature of the cAMP "receptor" is unknown. We show that extracts of the achlorophyllous ZC mutant of Euglena gracilis contain two cAMP-binding proteins, which bind cAMP with a high affinity (Kd values of 10 nM and 30 nM) and which can be separated by DEAE-cellulose chromatography. Protein kinase activity was assayed using Kemptide as a substrate. Stimulation of kinase activity by cAMP was observed after partial purification by DEAE-cellulose chromatography. Two peaks of activity were resolved, corresponding to distinct enzymes with different cAMP-analog specificities. Thus, cAMP signaling in plant cells may proceed by the phosphorylation of target proteins by cAMP-dependent kinases, in a manner similar to that of animal cells.

Oesophageal hiatus hernia in three generations of one family.

A family is described in which eight members in three successive generations had a sliding type of oesophageal hiatus hernia (or partial thoracic stomach) as a sole anomaly. The trait in this family behaved as a strict dominant, manifesting the segregation of a single abnormal autosomal gene. Other possible aetiologies are discussed but considered unlikely.

The molecular genetics of circadian rhythms in Arabidopsis.

While a number of physiological and biochemical processes in plants have been found to be regulated in a circadian manner, the mechanism underlying the circadian oscillator remains to be elucidated. Advances in the identification and characterization of components of the plant circadian system have been made largely through the use of genetics in Arabidopsis thaliana. Results so far indicate that the generation of rhythmicity by the Arabidopsis clock relies on molecular mechanisms that are similar to those described for other organisms, but that a totally different set of molecular components has been recruited to perform these functions.

Circadian rhythmicity in the activities of adenylate cyclase and phosphodiesterase in synchronously dividing and stationary-phase cultures of the achlorophyllous ZC mutant of Euglena gracilis.

Key factors in the adenosine 3',5'-cyclic monophosphate (cyclic AMP) metabolic pathway are two enzymes responsible for its generation and degradation, namely, adenylate cyclase (AC) and phosphodiesterase (PDE). In LD: 12,12 (12 h light, 12 h dark), these enzymes were found to undergo bimodal, circadian variation of activity in both dividing and nondividing cultures of the photosynthesis-deficient, achlorophyllous ZC mutant of Euglena gracilis Klebs (Z). Maximal AC activity occurred 2 h after the onset of the light interval (CT 02) and at the beginning of darkness (CT 12-14); these times corresponded to the acrophase profile for the rhythmic changes in cyclic AMP content that have been previously reported. The activity of PDE also exhibited a daily oscillation, but with an inverse phase pattern. Both the AC and PDE activity rhythms persisted after the cultures were transferred from LD: 12,12 to constant darkness. The activity of AC was activated significantly in vivo by forskolin at the trough phase (CT 20), while that of PDE was inhibited by 3-isobutyl-1-methyl-xanthine (IBMX) at its peak phase. These results indicate that the rhythms of both AC and PDE may be the main factors generating the circadian oscillations of cyclic AMP content in Euglena, which appear to be under control of an endogenous pacemaker.

Twenty to 40 year follow up of infantile hiatal hernia.

The aim of this study was to assess clinical and radiological findings of gastro-oesophageal reflux in adults who were diagnosed as having a hiatal hernia in infancy or early childhood. One hundred and eighteen patients with a minimum age of 20 who were diagnosed as having a hiatal hernia in childhood were interviewed; barium meal examination was performed in 96 of these cases. Ninety four patients had not required surgery for their hernia. The hiatal hernia persisted in 53% of these patients and 46% experienced heartburn at least monthly but in only three was this severe. Heartburn was significantly more common in patients in whom reflux was seen on barium meal. The consumption of antacids was significantly lower (20% v 46%) in patients who responded well to treatment as children. Eighteen of 24 patients who underwent surgery as children experienced heart-burn monthly but in only one patient was this severe. Two patients underwent endoscopy at their request because of symptoms during this follow up. Both had Barrett's oesophagus. In conclusion, despite the persistence of the hiatal hernia in half of the non-surgically treated patients, few complained of significant symptoms. Effective treatment in childhood was associated with a significant reduction in antacid consumption for heartburn as adults. The finding of Barrett's oesophagus in two patients high-lights a possible role for endoscopic screening in this patient group.

Familial hiatal hernia in a large five generation family confirming true autosomal dominant inheritance.

BACKGROUND: Familial hiatal hernia has only rarely been documented. AIMS: To describe the pattern of inheritance of familial hiatal hernia within an affected family. SUBJECTS: Thirty eight members of a family pedigree across five generations. METHODS: All family members were interviewed and investigated by barium meal for evidence of a hiatal hernia. RESULTS: Twenty three of 38 family members had radiological evidence of a hiatal hernia. No individual with a hiatal hernia was born to unaffected parents. In one case direct male to male transmission was shown. CONCLUSIONS: Familial inheritance of hiatal hernia does occur. Evidence of direct male to male transmission points to an autosomal dominant mode of inheritance.