Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.     

Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides,calcium, protein kinases and phosphatases and depolarization

Protein phosphorylation was investigated in [³²P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both ₁- and ₂-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the ₂-AR only marginally increased while the stimulation of ₁-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M₂-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated G_i-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of ₁-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported.     

Maintenance of transposable element copy number in natural populations of Drosophila melanogaster and D. simulans

To investigate the main forces controlling the containment of transposable elements (TE) in natural populations, we analyzed the copia, mdg1, and 412 elements in various populations of Drosophila melanogaster and D. simulans. A lower proportion of insertion sites on the X chromosome in comparison with the autosomes suggests that selection against the detrimental effects of TE insertions is the major force containing TE copies in populations of Drosophila. This selection effect hypothesis is strengthened by the absence of the negative correlation between recombination rate and TE copy number along the chromosomes, which was expected under the alternative ectopic exchange model (selection against the deleterious rearrangements promoted by recombination between TE insertions). A cline in 412 copy number in relation to latitude was observed among the natural populations of D. simulans, with very high numbers existing in some local populations (around 60 copies in a sample from Canberra, Australia). An apparent absence of selection effects in this Canberra sample and a value of transposition rate equal to 1–2 × 10^-3 whatever the population and its copy number agree with the idea of recent but temporarily drastic TE movements in local populations. The high values of transposition rate in D. simulans clearly disfavor the hypothesis that the low amount of transposable elements in this species could result from a low transposition rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Response of Douglas fir seedlings to phosphorus fertilization and influence of temperature on this response

Summary Effects of P fertilizers on growth of Douglas fir (Pseudotsuga menziesii var.menziesii (Mirb.) Franco.) seedlings were examined in pots and nursery beds. In pot experiments levels of P equivalent to 300 kg/ha were adequate for maximum growth over 14–18 weeks and resulted in available soil P levels of 80 ppm after 15 weeks' growth. Maximum growth in pots was obtained with shoot P concentrations of 0.18%–0.20%, with higher values at lower temperatures, but the optimum concentration for one-year old (1-0) nursery seedlings was 0.16% P. Growth of seedlings was greatly restricted at a soil temperature of 5°C and an air temperature of 12°C. At a soil temperature of 10°C and an air temperature of 14°C seedling P requirement was greater than at soil and air temperatures of 20°C.Comparison showed that monammonium phosphate was more effective than calcium superphosphate in stimulating growth in pots and nursery. Triple superphosphate was also effective in the nursery. Diammonium phosphate, potassium dihydrogen phosphate and phosphoric acid had no advantages as P sources in the nursery. Available P levels of 100–130 ppm, in the loamy sand and sandy loam nurseries studied, and needle P concentrations of 0.18%, when sampled in October, were associated with maximum growth of two-year old (2-0) seedlings.P fertilization decreased root/shoot ratio, but did not alter the allometric relationship of shoot to root. Improving P status from a low level increased root growth capacity in 2-0 seedlings and P fertilization of potted seedlings increased dry weight/height ratio. Uptakes per seed bed ha of 236 kg N, 31 kg P, 81 kg K and 73 kg Ca by 2-0 seedlings were comparable with, or greater than, uptake rates of agricultural crops. Recoveries of 6–11% of P from fertilizer were recorded in the nursery.     

Metabolic regulation in the facultative methylotroph Arthrobacter P1. Growth on primary amines as carbon and energy sources

During growth of the facultative methylotroph Arthrobacter P1 on methylamine or ethylamine both substrates are metabolized initially in an identical fashion, via the respective aldehydes. The regulatory mechanisms governing the synthesis and activities of enzymes involved in amine and aldehyde utilization were studied in substrate transition experiments. Transfer of ethylamine-grown cells into a medium with methylamine resulted in immediate exeretion of low levels of formaldehyde (max. 0.5 mM) and formate. In the reverse experiment, transfer of methylaminegrown cells into a medium with ethylamine, excretion of much higher levels of acetaldehyde (max. 3.5 mM) occurred. These different levels of aldehyde accumulation were also observed in studies with mutants of Arthrobacter P1 blocked in the synthesis of hexulose phosphate synthase or acetaldehyde dehydrogenase. In wild type Arthrobacter P1, aldehyde production resulted in rapid induction of the synthesis of enzymes involved in their degradation but also in temporary inhibition of further amine utilization and growth. The latter aetivities only resumed at normal rates after the disappearance of the aldehydes from the cultures. Acetaldehyde utilization resulted in intermittent excretion of ethanol and acetate, whereas formaldehyde utilization resulted in further accumulation of formate.During growth of Arthrobacter P1 in the presence of methylamine accumulation of toxic levels of formaldehyde is prevented because of the rapid synthesis of hexulose phosphate synthase to high activities and, in transient state situations, by feedback inhibition of formaldehyde on the activities of the methylamine transport system and amine oxidase.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoate) - HPS hexulosephosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Functional group contributions to the partitioning of phenols between liposomes and water

The temperature dependency of the partitioning of p-alkylphenols and p-halophenols has been determined between dimyristoyl phosphatidylcholine liposomes and 0.15 M NaCl. Partition coefficients increased as a function of temperature below the endothermic phase transition temperature (T_c) of the phospholipid but decreased above this temperature. The transfer process was found to be entropy-dominated below and enthalpy-dominated above the T_c, although large negative entropy changes were observed. Regular changes in the thermodynamic functions, partition coefficients and functional group free energies occurred as a function of the alkyl chain length or size of the halogen substituent below but not above the T_c. This has tentatively been attributed to increased phenol-phospholipid interaction at the higher temperatures. The partitioning of p-fluorophenol behaved in a manner expected of fluorinated compounds, yielding relatively low partition coefficients, but it produced an additional effect of markedly lowering the T_c of dimyristoyl phosphatidylcholine. Good correlations of the partition coefficients in liposomes with those in bulk organic solvents and with molecular size of the solute have been obtained.     

Effect of sodium butyrate on the hepatoma cell cycle: Possible use for cell synchronization

Summary Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse cinematography and [³H]thymidine incorporation studies. Evidence is presented that sodiunm butyrate affected the cell cycle at a specific point immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used for HTC synchronization. This work was supported by l'Institut Nationale de la Santé et de la Recherche Médicale and la Centre Nationale de la Recherche Scientifique (L. T. and J. K.).     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase     

Yearly maps of summer temperatures in Western Europe from A.D. 1750 to 1975 and Western North America from 1600 to 1982:

The primary purpose of this publication is to make available two series of historical maps showing summer (April–September mean) temperatures over western Europe from A.D. 1750 and western North America from A.D. 1600. These maps have been reconstructed using networks of temperature-sensitive tree-ring chronologies as part of a continuing collaboration between the Swiss Federal Institute for Forest, Snow and Landscape Research (FSL) at Birmensdorf, Switzerland and the Climatic Research Unit (CRU) at the University of East Anglia, Norwich, U.K. The maps were reconstructed using relatively long maximum latewood density chronologies selected from the extensive networks of tree-ring data produced at the FSL. The reconstructions were produced using a spatial regression technique developed at CRU.The basis for the reconstructions are wood samples from various conifers growing at cool-moist sites close to the alpine or boreal timberlines. This material was analysed by x-ray densitometry and the resulting maximum latewood density chronologies were calibrated against meteorological data over a 100-year calibration period.These results emphasize the value of dendroclimatological research which can provide continentalscale reconstructions of past climate which represent a potential resource for climatologists, biologists, and historians with an interest in the spatial variability of summer temperatures over the middle to high latitudes.