稀土离子对钙调蛋白与单克隆抗体分子识别的影响

分别采用酶联免疫吸附(ELISA)法和荧光标记技术比较了Ca²⁺,La³⁺,Eu³⁺和Yb³⁺离子对钙调蛋白与单克隆抗体2C3之间分子识别的影响.结果表明,金属离子与钙调蛋白作用后会诱导其发生不同的构象变化,并进一步影响到钙调蛋白与单克隆抗体2C3分子之间的结合强度.当钙调蛋白分别与La³⁺,Eu³⁺,Yb³⁺作用后,它与单抗2C3分子之间的解离常数为(26.8±2.5),(21.8±3.4)和(64.8±5.1)nmol/L,而结合Ca²⁺前后的钙调蛋白与单抗分子的解离常数分别为(177.2±2.8)和(157±4.2)nmol/L.这一结果表明,稀土离子诱导钙调蛋白发生的构象变化明显不同于钙离子的作用,这种差异可能是稀土与钙离子对钙调蛋白调控作用表现出差别的原因.     

INFLUENCE DE LA LUMIÈRE SUR LES MOUVEMENTS DE NUTATION DES PLANTULES

Abstract— Red light can promote (a) the straightening of the curvature ( Phaseolus vulgaris, Pisum sativum ) (b) the formation of a loop ( Phacelia tanacetifolia and Picea abies ) or (c) the induction of a curvature which does not occur in the darkness ( Lactuca sativa ). Thesc red-light induced movements could be reversed by following each brief red exposure with short irradiation by far-red light. These reactions are controlled by the phytochrome system.
On the other hand, exposure to far-red and blue light during a long period can induce other movements:
(a) The light re-opens the hook which has been closed by red radiation ( Lactuca sativa ).
(b) The light induces the straightening of the dark-induced curvature ( Phacelia tanacetifolia ). These facts may be due to an unknown property of phytochrome or to the action of another photoreceptor.     

Heavy metal immobilization in aqueous solution using calcium phosphate and calcium hydrogen phosphates

This study examines the possibilities for removing heavy metal cations from water with calcium phosphate, calcium hydrogen phosphate, and calcium dihydrogen phosphate at 293 K. It was reported that immobilization of aqueous heavy metal cations, which is known to be one of the characteristic properties of calcium hydroxyapatite, proceeded favorably with these phosphates. Calcium phosphate, calcium hydrogen phosphate, and calcium dihydrogen phosphate could favorably remove Pb2+ from aqueous solution. Calcium hydrogen phosphate also removed aqueous Cu2+, Co2+, and Cd2+, whereas these cations were not immobilized by calcium phosphate and calcium dihydrogen phosphate. A contribution of the dissolution-precipitation mechanism to immobilization with these phosphates is suggested.     

Light Effects on Cyclic Nucleotide Levels and Phase Shifting of the Circadian Clock in Neurospora crassa

Abstract— Mycelia of Neurospora crassa (band [bd] mutant) were exposed to white light (blue light intensity 3.5 μ.mol s^?1 m^?2) of different durations during constant darkness. The concentrations of different second messenger molecules and the phase shifting of the circadian rhythm were determined during light exposures at circadian time (ct) 12 and thereafter. These light exposures elicited 8-12 ct units delay phase shifts but did not change the amount of inositol 1,4,5-triphosphate (InsP₃). In contrast, significant effects of light were observed on cyclic adenosine 3′,5′-monophosphate (cAMP) levels, which increased transiently about 30-90 s after the onset of light. The same kinetics was observed under continued exposure to light as well as after 10 s and 2 s of light followed by darkness. The relative amount of the cAMP-dependent protein kinase A (PKA) in the form of its catalytic sub-units was determined in isolated nuclei of the bd strain in relation to total nuclear proteins by means of western blot analysis, using a heterologous antibody. The nuclear PKA content changed parallel to the cAMP changes. The transient increase of cAMP did not occur in the “blind”white collar mutant (wc-2). Long-term kinetics of cAMP changes after different light pulses in bd showed the initial increase and a return to control levels about 10 min after the onset of the light pulse and a subsequent longer lasting decrease. Light-induced cAMP changes and light-induced phase shifts showed different duration dependencies, thus indicating that cAMP may not play a role in the signal transduction pathway to the clock. Light exposures, furthermore, led to a significant decrease of the cyclic guanosine 3′,5′-monophosphate (cGMP) level. Long-term kinetics of the cGMP content again showed the immediate decrease after 2 min and a slow recovery to (or above) control values after several hours. Various calcium channel blockers (nickel, cobalt, nifedipine, dantrolene, lanthanum) and the calcium calmodulin inhibitor chlorpromazine did not affect the phase shifting by light nor did they significantly phase shift the circadian rhythm in the dark themselves (again determined at ct 12). The data showed that InsP₃ did not change after exposure to light, whereas cAMP increased and cGMP decreased. There was no evidence, however, that these changes play a role in light signal transmission to the clock.     

BLUE and NEAR ULTRAVIOLET REVERSIBLE PHOTOREACTION IN THE INDUCTION OF FUNGAL CONIDIATION

Abstract— The effects of blue light and near UV light on the induction of conidiation in the fungus, Aliernuriu tomato, were investigated. Induction of conidiation was repeatedly controlled by alternating doses of near UV light and blue light. When the final light was near UV, conidiation was induced and conidia developed in the following darkness; when it was blue, the induction of conidiation was suppressed. When conidiation was induced by irradiation with a light mixed with near UV and blue, not only the time lag for inducing conidiation but also the amount of conidia formed were regulated by the fluence rates of both those lights.
Thus, 'mycochrome' is considered to function as a photoreceptor system in the induction of conidiation of this fungus.     

PHOTOCONTROL OF HYPOCOTYL ELONGATION IN DE-ETIOLATED Cucumis sativus L. RAPID RESPONSES TO BLUE LIGHT

Abstract— Rapid inhibition of hypocotyl extension of de-etiolated Cucumis sativus L. by blue light is described, and compared with responses to white fluorescent light. Rapid inhibition of hypocotyl growth by blue light via the specific blue light photoreceptor requires a minimum Buence rate. Above this minimum value rapid modulations of growth rate accompany changes in blue light fluence rate. An initial response to blue light, or to a step-up in blue fluence rate takes less than 5 min. A drop from high to low fluence rate blue light (or to darkness) is followed by a recovery of the growth rate after about 20 min. A change from a low fluence rate to darkness elicits a more rapid recovery of growth rate (within 7 min). Similar responses were obtained in seedlings de-etiolated for a few hours and for several days.     

A POSSIBLE CONTROL BY PHYTOCHROME and OTHER PHOTORECEPTORS OF PROTEIN ACCUMULATION IN THE GREEN ALGA Ulva rigida

Abstract— The effects of red, blue and green light pulses on total protein accumulation in the green alga Ulva rigida following transfer from a low to high nitrate medium in darkness were examined. Red light pulses prior to transfer to darkness increased protein accumulation by about 55%. Blue and green light pulses also stimulated protein accumulation, but to a lesser extent (40-30% respectively). Stimulation of protein accumulation by red, blue or green light was largely (red light) or partially (blue or green light) reversible by far-red. The role of phytochrome and a red/green photoreversible system in the control of protein synthesis is discussed.     

Distinct responses of chloroplasts to blue and green laser microbeam irradiations in the centric diatom Pleurosira laevis

The centric diatom Pleurosira laevis is a large unicellular alga, in which ca 200 chloroplasts migrate toward the nuclear cytoplasm through the transvacuolar cytoplasmic strands in response to blue-light irradiation and, on the contrary, toward the cortical cytoplasm in response to green-light irradiation. We analyzed these light-induced chloroplast migrations using a scanning laser microbeam provided by a confocal microscope for intracellular irradiation. Spot irradiation of a blue laser microbeam induced rapid assemblage of chroloplasts into the nuclear cytoplasm regardless of the spot position and spot number. On the other hand, one or two spots of green laser microbeam induced chloroplast accumulation at the spots, although increasing spot numbers suppressed chloroplast accumulation at each spot. In our experimental condition, ca 1 min of blue-light irradiation was sufficient to stimulate movement, whereas green-light irradiation required uninterrupted and longer irradiation time (ca 15 min). Chloroplast assemblage induced by blue-light required extracellular Ca2+, and was inhibited by Ca2+ channel antagonists. Furthermore, higher efficiencies of chloroplast migration were obtained when a single beam spot was fragmented and scattered over wider area of plasma membrane. These observations suggested that blue-light induced a response at the plasma membrane, which subsequently activated Ca2+ permeable channels. This sequence of physiological events is identical to what was previously observed with chloroplast movement in response to mechanical stimulation. Furthermore, experiments with the cytoskeleton-disrupting agents, colchicine and cytochalasin D, indicated that blue-light-induced chloroplast movement required microtubules whereas the green-light-induced response to beam spot required actin filaments.     

Effects of Calmodulin Inhibitors and Blue Light on Rhythmic Movement of Robinia pseudoacacia Leaflets

Abstract— The effect of several calmodulin (CAM) antagonists, blue light and an intracellular calcium inhibitor, on the circadian rhythm of Robinia pseudoacacia leaflet movement has been studied. The CAM antagonists, chlorpromazine (CPZ), trifluoperazine (TFP), calmidazolium and N -(6-aminohexyI)-5-chloro-1-naphthalenesulfonamide (W₇) shifted the phase of the circadian rhythmic movement while W₅, an inactive analogue of W₇, had no effect. Two hour pulses of calmidazolium (10–50 μ M ) gave rise to a phase-response curve with maximum advances (up to 9 h) at circadian time (CT) 6 and maximum delays (up to 7 h) at CT 22. No effect was found on transition from subjective day to subjective night and vice versa. The TFP (10–50 μ M ), applied as 2 h pulses during the circadian cycle, shifted the phase of the circadian leaflet movement and also produced maximum advances in the middle of subjective day. Two hour blue light pulses shifted the phase of leaflet rhythmic movement. The phase-response curve obtained showed maximum advances (up to 5 h) in the middle of subjective day and maximum delays on transition from subjective day to subjective night. Two hour pulses of 50 μ M 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hypochloride (TMB-8), an intracellular calcium inhibitor, caused the same type of phase-response curve, with maximum advances and delays occurring at the same time as those produced by blue light. These results indicate that CAM might be involved in controlling the circadian oscillator that drives Robinia leaflet movement. The relationship between CAM and calcium with red and blue light is discussed.     

Electrospray ionization mass spectrometry and hydrogen/deuterium exchange for probing the interaction of calmodulin with calcium

The extent of H/D exchange of the protein calmodulin in solution was monitored by mass spectrometry following electrospray ionization (ESI) of the protein. In the absence of Ca2+, approximately 115 protons are exchanged for deuteriums after 60 min. As the calmodulin is titrated with Ca2+, the extent of exchange decreases significantly (i.e., by 24 protons), indicating Ca(2+)-induced folding of the protein to a tighter, less solvent-accessible form. The extent of H/D exchange ceases to decrease when the amount of added Ca2+ is sufficient to convert greater than 80% of the calmodulin to a form bound by four calcium ions. Lysozyme, a protein of similar molecular weight, does not show a significant decrease in the extent of H/D exchange as it binds to Ca2+, indicating that the changes in H/D exchange for calmodulin reflect tertiary structural change that occur upon binding with Ca2+.     

PHOTOINHIBITION OF GROWTH OF- A YELLOW AND A COLORLESS FORM OF CHLAMYDOMONAS REINHARDTII*

Abstract— Long-term growth on acetate in darkness of a permanently yellow and a white-in-darkness mutant of Chlamydomonus reinhardtii showed no requirement for light. Room light or moderate intensities of white light severely inhibited the white mutant, but allowed continuous growth of the yellow form. Blue light (460 nm) stops motility and growth, and induces clumping, in the white form at energy levels which only reduce the growth rate of the yellow mutant. Green light (550 nm) had no effect on either strain, while red light (630 nm), at equal energy levels, affected the white mutant only, however, not as severely as blue light. The mechanism of action of blue and red light is unknown. Evidence suggested the inhibition is not due to cell exudates.     

INFLUENCE DES ÉCLAIREMENTS APPROXIMATIVEMENT MONOCHROMATIQUES SUR LE DEVELOPPEMENT DES TISSUS DE MOELLE DE TABAC CULTIVBS IN VZTRO EN PRESENCE D'AUXINE ET DE KINETINE

Abstract— The influence of relatively monochromatic light (around 4350, 5250, 6600 and 8000 Å) on the growth of cultures of tobacco pith tissues with auxin and kinetin was studied. The cultures were subjected either to continued light or to alternations of 16 hr light with 8 hr darkness, or to a dark period followed by continuous light.
The influence of blue light was especially noted. The development in volume and weight of the cultivated tissues in blue light is generally less than in the other conditions (except for one or two exceptions); the differences are attenuated when a light period alternates with a period of darkness; the callus tissues of tobacco pith exposed to blue light are always chlorophyllic with a dense and hard appearance and histological studies show that tissues grown in blue light are mostly made of large cells with thick cell-walls and many nodules while under the other conditions the tissues are mostly made of very small cells with few or no nodules.
These differences seem related to specific photoreceptors rather than to energies delivered to the cultures.     

Effects of Selenium and Light Wavelengths on Liquid Culture of <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Link

To investigate the effects of selenium and light wavelengths on the growth of liquid-cultured Cordyceps militaris and the main active components’ accumulation, culture conditions as selenium selenite concentrations and light of different wavelengths were studied. The results are: adenosine accumulation proved to be significantly selenium dependent (R ² = 0.9403) and cordycepin contents were determined to be not significantly selenium dependent (R ² = 0.3845) but significantly enhanced by selenium except for 20 ppm; there were significant differences in cordycepin contents, adenosine contents, and mycelium growth caused by light wavelengths: cordycepin, blue light > pink light > daylight, darkness, red light; adenosine, red light > pink light, darkness, daylight, blue light; and mycelium growth, red light > pink light, darkness, daylight > blue light. In conclusion, light wavelength had a significant influence on production of mycelia, adenosine, and cordycepin, so lightening wavelength should be changed according to target products in the liquid culture of C. militaris.     

CIRCADIAN RHYTHM OF Robinia pseudoacacia LEAFLET MOVEMENTS: ROLE OF CALCIUM AND PHYTOCHROME

Abstract— The effect of external calcium level, calcium ionophore A23187 and red light on the circadian rhythm of Robinia pseudoacacia leaflet movements has been studied. Fifteen minute red light pulses shifted the phase of leaflet rhythmic movement with a phase-response curve type 0. Maximum advances and delays (about 10 h and 8 h, respectively) were obtained between circadian time (CT) 10 and CT 12 at the end of a subjective day. An almost null effect was obtained at the end of a subjective night. Phytochrome is the photoreceptor involved in phase shifting since this effect of red light is reversed by 5 min of far red light. Two hour pulses of external calcium, applied as CaCl₂ (10 m M ), and 2 h pulses of calcium ionophore A23187 (10–50 μM) also shifted the phase of leaflet circadian movement and caused the same type of phase-response curve, with maximum advances and delays at the same time as those produced by red light. Two hour pulses of an external calcium chelator, EGTA (5 m M ), and a calcium channel blocker, LaCl₃ (10–50 m M ), damped the circadian rhythm or did not change the phase when they were applied at lower concentration. These results indicate that phytochrome could control the circadian oscillator, which drives Robinia leaflet movements by increasing the intracellular calcium concentration.     

Nanosecond electric pulse-induced calcium entry into chromaffin cells

Electrically excitable bovine adrenal chromaffin cells were exposed to nanosecond duration electric pulses at field intensities ranging from 2 MV/m to 8 MV/m and intracellular calcium levels ([Ca(2+)](i)) monitored in real time by fluorescence imaging of cells loaded with Calcium Green. A single 4 ns, 8 MV/m pulse produced a rapid, short-lived increase in [Ca(2+)](i), with the magnitude of the calcium response depending on the intensity of the electric field. Multiple pulses failed to produce a greater calcium response than a single pulse, and a short refractory period was required between pulses before another maximal increase in [Ca(2+)](i) could be triggered. The pulse-induced rise in [Ca(2+)](i) was not affected by depleting intracellular calcium stores with caffeine or thapsigargin but was completely prevented by the presence of EGTA, Co(2+), or the L-type calcium channel blocker nitrendipine in the extracellular medium. Thus, a single nanosecond pulse is sufficient to elicit a rise in [Ca(2+)](i) that involves entry of calcium via L-type calcium channels.     

QUELQUES OBSERVATIONS SUR LE DÈVELOPPEMENT DES CULTURES IN VITRO DE MOELLE DE TABAC EN PRÉSENCE D'AUXINE ET DE KINÉTINE, EN ÉCLAIRE-MENT RELATIVEMENT MONOCHROMATlQUE

Abstract— In a previous publication we have presented some results on tobacco pith tissue cultures grown in relatively monochromatic light (around 4350,5250, 6600 and 8000 Å).
This paper points out specially that cultures subjected to a continuous blue light period of 12 days only, followed by a darkness period of 29 days, grew no more than cultures exposed to the same radiations during the 41-day period of the experiment.
The growth was only 67 per cent of that of tissues grown in the darkness.
The morphologic and histologic observations showed the same differences between the cultures grown in blue light conditions and the cultures grown in the other light conditions, as also reported elsewhere.
Observation of bud developement on stem segment cultures in vitro of willow, grown under these relatively monochromatic light conditions, together with these histologic studies, leads us to believe in a possible gibberelline action in blue light.     

POSSIBLE ROLES OF CALCIUM and CALMODULIN IN THE LIGHT-STIMULATION OF WALL BIOSYNTHESIS IN Phycomyces

Abstract— We studied the effect of light on the activity of chitin synthetase from Phycomyces blakeslee-anus in vitro in order to gain information on the biochemical bases of the photostimulation of sporangiophore growth. We observed that chitin synthetase activity of cell-free extracts prepared from both sporangiophores and vegetative mycelium was increased by illumination. The process was stimulated by calcium and was not affected by protease inhibitors. Iodoacetamide inhibited chitin synthetase activation by light, or by controlled proteolysis. Calcium activated chitin synthetase by two mechanisms, one independent of and one dependent on calmodulin, the latter being sensitive to trifluoperazine. Trifluoperazine also inhibits sporangiophore growth and more noticeably phototro-pism. We suggest that illumination increases net wall growth through two light-sensitive calcium and calcium-calmodulin systems one of which stimulates the normal chitin synthetase activation mechanism, and another one probably involved in vesicle displacement to the growing zone of the cell.     

THE PHOTOBIOLOGY OF Paphiopedilum STOMATA: OPENING UNDER BLUE BUT NOT RED LIGHT

Abstract— The responses of stomata from Paphiopedilum harrisianum , Orchidaceae, to light and CO₂ were studied in epidermal peels. Stomatal opening under red light was indistinguishable from that in darkness, whereas blue light promoted opening above dark levels. The ineffectiveness of red light in causing stomatal opening was confirmed in the presence of 100 μ M KCN; average apertures in both darkness and red light were 53% of those measured in the absence of the inhibitor, whereas under blue irradiation, the KCN inhibition was only 30%, with average apertures two-fold of those measured under red light or darkness. Fluence rate response curves under blue light were typical of a single photoreceptor; removal of CO₂ increased aperture values without a significant light-CO₂ interaction. The lack of a stomatal red light response contrasts with results obtained in species with chlorophyllous stomata in which red light consistently causes stomatal opening, and suggests that the previously reported red light responses in stomata from intact Paphiopedilum leaves resulted from indirect effects, such as depletion of intercellular CO₂ by mesophyll photosynthesis. In isolation, Paphiopedilum stomata appear to rely on a blue light photosystem for their responses to light and fail to open under red light because of their lack of guard cell chloroplasts.     

PHOTOCONTROL OF HYPOCOTYL ELONGATION IN DE-ETIOLATED Cucumis sativus L. A BLUE-LIGHT-INDUCED POST-ILLUMINATION BURST OF GROWTH

Abstract— A rapid transient increase in the growth rate of Cucumis sativus L. seedlings is found to occur after a transition from fluorescent white light to darkness. The post-illumination burst of growth starts after about 20 min of darkness and lasts for 30 to 50 min. The response occurs after long periods of continuous white light, in cycles of white light and darkness, whether the cotyledons are covered or exposed. The post-illumination burst is an expression of the loss of photocontrol of hypocotyl inhibition via the specific blue light photoreceptor because (a) its kinetics resemble those of recovery from blue-light inhibition, and (b) it only occurs on transition from a blue-rich to a blue-poor light environment.     

Melanopsin triggers the release of internal calcium stores in response to light

Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling.