PHOTOMORPHOGENESIS IN Penicillium isariaeforme: EXOGENOUS CALCIUM SUBSTITUTES FOR LIGHT

Abstract— Penicillium isariaeforme is a photomorphogenic fungus which produces upright bundles of conidia-bearing mycelia (called coremia) when grown on a defined medium in visible (450–500 nm) light. We found that exogenous Ca²⁺ ions could substitute for light. In the dark 1–2 m M Ca²⁺ triggered coremia formation. Dark induction of coremia was specific for Ca²⁺ in that it could not be duplicated by 50 m M Ba²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Sr²⁺, or Zn²⁺. Additionally, light-induced coremia formation was inhibited by both KI (2.5 m M ) and phenylacetic acid (0.25 m M ).     

PHOTOPHYSIOLOGY OF TURION GERMINATION IN Spirodela polyrhiza (L.) SCHLEIDEN–V. DEMONSTRATION OF A CALCIUM-REQUIRING PHASE DURING PHYTOCHROME-MEDIATED GERMINATION

Abstract— Etiolated turions of Spirodela polyrhiza are positively photoblastic and show a phytochrome-mediated low fiuence germination response. The far-red light (FR) reversibility decreased with the delay of FR irradiation (lag phase 1.06 ± 0.03 days after red light irradiation; half-maximal response 1.9 days). The action of the far-red-absorbing form of phytochrome (Pfr) was only realized by a germination response if exogenously applied Ca²+ was present. Calcium step-down (from 1 mM to 0.9 μ M Ca²+) and Ca²+ step-up (from 0.9 μ M to 1 m M Ca²+) experiments were carried out to determine the Ca²+-sensitive phase. There was no time gap between the two phases determined by the step-down and step-up experiments but a clear coincidence of both curves. Pulse treatments (24 h) with Ca²+ (1 m M ) showed the upper part of this common curve to represent the most Ca²+-sensitive phase. The Ca²+-sensitive phase was within the Pfr-requiring phase. After reversion of Pfr by FR pulses there was only a negligible response to the high Ca²+-concentration, independent of the delay between the red light (R) and FR pulses. These results are compatible with the assumption of Ca²+ acting as a second messenger of Pfr. However, the Ca²+-insensitivity in the first 12 h after the R pulse points against this hypothesis.     

CALCIUM CONTROL OF PHOTOTACTIC ORIENTATION IN Chlamydomonas reinhardtii: SIGN AND STRENGTH OF RESPONSE

Abstract— Chlamydomonas reinhardtii responds to a blue light stimulus by an oriented swimming (phototaxis) toward or away from the stimulus source. In this study it is established that the sign and strength of the phototactic response are a complex function of extracellular [Ca²⁺], stimulus fluence rate, time of analysis after onset of stimulation and light pretreatment. At very low extracellular [Ca²⁺] the response is weak and usually negative. At [Ca²⁺] close to the preconditioning level, phototactic response becomes stronger and positive. As [Ca²⁺] is raised further, the initial (2 s) response remains positive but the long term (20 s) becomes negative and very strong. At extremely high [Ca²⁺] the cells become immobile. This bimodal behavior suggests that two different mechanisms determine the direction of the turn. Data cannot be explained in terms of a simple model. The model which accounts for most of the details of the behavior is that of Kamiya and Witman (1984), which proposes that positive response is triggered by a transient increase in intracellular [Ca²⁺] and negative response by a decrease below unstimulated level of Ca²⁺, at least in the range of 10^-9-10^-6 M [Ca²⁺]. The strong negative orientation which follows an initial positive response above this level of [Ca²⁺], in these experiments, is best explained by an adaptation of the cells due to an increased (on average) intracellular [Ca²⁺].     

INDUCTION OF LEAF UNROLLING BY PHYTOCHROME and ACETYLCHOLINE IN ETIOLATED WHEAT SEEDLINGS

Abstract-Phytochrome regulates the unrolling of primary leaf sections from 8-day-old dark-grown wheat ( Triticum aeslivum L. cv. Arminda) seedlings. Red light (R)-stimulated unrolling of leaf sections pretreated in 1 m M ethylene-bis-(β-aminoethylether)- N,N,N',N' -tetraacetic acid (EGTA) if 1 m M CaCl₂ was added during a 30 min treatment period including and following irradiation. Nifedipine at 1 μ M (a Ca²+-channel antagonist) applied 10 min before R prevented the R stimulation of leaf unrolling. The Ca²+-channel agonist Bay K-8644 (1 μ M ) and acetylcholine (ACh, 1 mY M ) stimulated unrolling of leaf sections prewashed in EGTA in darkness, if 1 m M CaCl₂ was present in the medium during a 30 min treatment period. Acetylcholine also induced leaf unrolling in the absence of Ca²+ when 100 μ M NaCl was present in the medium. Apart from ACh, only carbamylcholine out of the choline derivatives tested was active in induction of leaf unrolling in the presence of 1 m M Ca²+. The ACh receptor antagonists, atropine (10 μ M ) AND D-tubocurarine (10 μ M ), nullified the ACh-induced Ca²+- and Na+-dependent leaf unrolling, respectively. Muscarine and nicotine, agonists of ACh, at 1 μ M stimulated leaf unrolling in the presence of Ca²+ and Na+, respectively. The ACh-induced Ca²+-dependent leaf unrolling was reduced by 1 μ M Nifedipine, 10 μ M Li+ and 10 μ M "calmodulin" inhibitor, trifluoperazine (TFP), whereas only TFP was active in the reduction of the Na+-dependent ACh-induced leaf unrolling response. It is proposed that leaf unrolling of dark-grown primary wheat leaves can be regulated by phytochrome and by activation of two different types of ACh receptors.     

THE REGENERATION OF VISUAL PIGMENTS AND THE CHANGE OF ROD HYPERSENSITIVITY AFTER IRRADIATION BY BLEACHING LIGHT IN FROG RETINA

Abstract— The regeneration processes of visual pigments and the dark adaptation processes of rod photoreceptor after irradiation by bleaching light were studied by spectrophotometric, electroretinographic(ERG) methods and the measurement of early receptor potentials (ERPs) in bullfrog retina. After irradiation by bleaching light, rhodopsin in the isolated retina regenerated to an extent depending on the wavelength and intensity of the bleaching light as well as pH. Intense blue light and a weak alkaline environment (pH 7.5–9.5) favoured the regeneration. The regeneration of pigment in the green rods could not be detected in these experiments on the isolated retina. The regeneration of cone pigment was studied by measuring ERPs from both isolated retinas and retinas with pigment epithelium-choroid complex separated from scleras, which are called PEC-retinas. In the PEC-retinas, cone pigment regenerated more rapidly and with better efficiency than in the isolated retinas.
Rod photoreceptors desensitized permanently by bleaching light did not demonstrate hypersensitivity at 0.1 m M [Ca²⁺]_out, which induced hypersensitivity in non-desensitized photoreceptor, but showed the hypersensitivity when the [Ca²⁺]_out, was lowered further by the addition of EGTA.     

EFFECTS OF K+ AND Ca2+ IONS ON MOTILITY AND PHOTOSENSORY RESPONSES OF STENTOR COERULEUS

Extracellular K⁺ ions above a critical concentration induce ciliary reversal in unstimulated Stentor coeruleus and suppress step-up photophobic response. This threshold concentration of K⁺ ions depends on the extracellular Ca²⁺ concentration, and the subsequent backward gyration and light-sensitivity suppression seem to depend on the relative concentrations of K⁺ and Ca²⁺. The concentration of Ca²⁺ necessary to overcome K⁺-mediated inhibition of phobic response and backward swimming increases non-linearly with increasing K⁺ concentration. The Ca²⁺-blocking agent. D-600, selectively inhibits photophobic responses of Stentor , thus further confirming the role of Ca²⁺ ions in photosensory transduction of this ciliate.     

PHTHALOCYANINE PHOTOSENSITIZATION OF MAMMALIAN CELLS: BIOCHEMICAL and ULTRASTRUCTURAL EFFECTS

Abstract— The concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺ and CI⁻ ions in the cytoplasm of octopus photoreceptor cells were determined before and after illumination by electron probe X-ray microanalysis. The concentrations of these elements in the dark-adapted photoreceptor cells were: Na⁺, 68.4; K⁺, 111.4; Ca²⁺, 4.0; Mg²⁺, 16.4; and CI⁻, 102.9 m M /kg of cytoplasm. Illumination raised the concentration of Na⁺ by 58 m M and that of Cl⁻ by 23 m M and reduced the K⁺ concentration by 47 m M /kg of cytoplasm. A trace increase of intracellular Ca²⁺ and a trace decrease of Mg²⁺ were observed. These results confirm the hypothesis that sodium ions flow in on illumination, and suggest the influx of chloride ions and the outflux of potassium ions during illumination. The intracellular concentrations of Na⁺, K⁺ and Cl⁺ can give the basis for calculating the ion permeability of ion channels in octopus photoreceptor cell membranes, using values of the membrane potentials obtained by electrophysiological studies     

SALT AND pH-DEPENDENT CHANGES OF THE PURPLE MEMBRANE ABSORPTION SPECTRUM

Abstract —Purple membrane suspensions change their color to blue and the absorption maximum shifts to 608 nm when the membrane is deionized on a cation exchange column or when it is washed first with < 2N NaCl followed by deionized water. The deionized chromophore is essentially identical with the chromophore produced by lowering the pH of the native membrane to < 4.0 (p K < 3.0). However, the deionized membrane does not aggregate and can be obtained in the pure state. The original purple color of the membrane is restored by addition of around 1 m M Na⁺, K⁺ or 10 μ M Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Pb²⁺ or La²⁺ when the protein concentration is 5μ M . The required salt concentrations decrease with decreasing pH. Direct measurement of bound Ca²⁺ by atomic absorption spectroscopy yields a ratio of Ca²⁺ to protein of <2 and a binding constant of 1.4 × 10⁶. Titration of the spectral change with salts at different pH values shows a linear relation between the pH and the logarithm of the salt concentration, with a 1:1 ratio for Na⁺ and 1:2 ratio for Ca²⁺. These relations are well predicted by Gouy-Chapman theory; however, the accompanying release of protons, changes of the CD spectrum, the complex kinetics of the spectral change during reconstitution with salt and preliminary X-ray diffraction results all suggest that conformational changes may be occurring in the protein.     

EVIDENCE FOR A MAGNESIUM DEPENDENT ATPase IN BOVINE ROD OUTER SEGMENT DISK MEMBRANES*†

Abstract—In the presence of Mg²⁺ and adenosine triphosphate (ATP), a rapid. light-induced, light-scattering transient is observed from bovine rod outer segments (ROS). This light-scattering transient we have labelled 'A'. Ca²⁺ cannot replace Mg²⁺. nor can guanosine triphosphate (GTP) replace ATP. 'A' is observed at ATP concentrations as low as a few μM.
The half-time of 'A', 60 ms at 20° and 20 ms at 37°, is consistent with a process possibly involved in visual transduction.
'A' has the action spectrum of rhodopsin bleaching and its amplitude is strictly proportional to the fraction of rhodopsin bleached per flash. 'A' can be regenerated by 11- cis retinal.
Inhibition studics with ATP analogues, which cannot be hydrolysed and fail to evoke an 'A' response, reveal that an ATP hydrolysis process has to precede illumination in order for 'A' to occur.
On the basis of the above findings. it is proposed that there is a Mg²⁺ dependent ATPase in ROS that allows the disk membrane to assume a new membrane state which, upon illumination, is altered. giving rise to the structural phenomenon monitored as light-scattering transient 'A'.     

EFFECTS OF IONOPHORES AND RUTHENIUM RED ON THE PHOTOTAXIS OF STENTOR COERULEUS AS MEASURED BY SIMPLE DEVICES

Abstract— Two simple methods of phototaxis measurements have been applied to study the effects of ionophores on the negative phototactic response in Stentor coeruleus. The inhibitory effects of Ca²⁺-ionophore (A23187), Ca²⁺-blocking agent (Ruthenium Red), and K⁺ -ionophore (valinomycin) on photo-taxis have been determined. Results suggest that the influx of Ca²⁺ plays a transducing role in the phototaxis of Stentor.     

Ca2+ INFLUX INDUCED BY PHOTODYNAMIC ACTION IN HUMAN CEREBRAL GLIOMA (U-87 MG) CELLS: POSSIBLE INVOLVEMENT OF A CALCIUM CHANNEL

Abstract The plasma membrane has been implicated as a critical target of photodynamic action on cells. We have observed that the photosensitization of human cerebral glioma (U-87 MG) cells by hematoporphyrin derivative (HpD) causes a large increase in intracellular calcium [Ca²⁺]. This increase in [Ca²⁺]_i was solely due to the influx of extracellular Ca²⁺ through the plasma membrane and showed a dependence on HpD concentration, light dose and concentration of calcium in the extracellular medium. The magnitude of the Ca²⁺ influx decreased with increasing postirradiation time, which suggests that the cell membrane partially recovers from the photodynamic injury. The photoinduced Ca²⁺ influx was inhibited by the Ca²⁺ channel blocker diltiazem and the reducing agent dithioerythritol. These findings are discussed in terms of possible activation of a Ca²⁺ channel as a result of photosensitization.     

HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN®) PHOTODYNAMIC ACTION ON Ca2+ TRANSPORT IN MONKEY KIDNEY CELLS (CV-1)

Abstract After 24 h incubation with Photofrinm^® (PF), photodynamic action has been studied on Ca²⁺ transport in CV-1 cells. A moderate increase of the cytosolic free Ca²⁺ concentration [Ca²⁺] is observed immediately after a dose of irradiation which yields a survival rate of less than 5% at 48 h. In parallel, studies on digitonin-permeabilized cells indicate that such a treatment inhibits endoplasmic reticulum Ca²⁺ uptake with few alterations of this process in mitochondria. In contrast, ADP-stimulated respiration is impeded and intracellular ATP level decreases. It is suggested that direct damage to endoplasmic reticulum as well as mitochondrial disturbance are the primary mechanisms responsible for a nontransient elevation of [Ca^2+]i preceding cell death.     

PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

ON THE SIGNAL-TRANSDUCTION CHAINS OF TWO Pfr- MEDIATED SHORT-TERM PROCESSES: INCREASE OF ANCHORAGE and MOVEMENT OF Mougeotia CHLOROPLASTS

Abstract— We examined two published hypotheses on the signal-transduction chain of the light-oriented chloroplast movements in the fresh-water alga Mougeotia. One hypothesis postulates a Ca²+-influx controlled by a tetrapolar gradient of phytochrome in its far-red light absorbing form (Pfr). The other hypothesis postulates anchorage sites for actin-filaments even at those areas of the plasmalemma where phytochrome is in its inactive form (Pr). Calmodulin and Ca²+-sequestering vesicles are assumed to be essential links of this transduction chain.
To test these hypotheses we have studied the effects of Ca²+-entry blockers, Ca²+ deficiency and calmodulin antagonists on chloroplast movements and on chloroplast anchorage. None of our results support the Ca²+/calmodulin hypotheses mentioned above. The results and their implications (with regard to the role of Ca²', calmodulin and anchorage sites) are discussed.     

PROTOCHLOROPHYLL(IDE)630 PHOTOSENSITIZES ACTIVE Ca2+ ACCUMULATION IN MICROSOMAL AND MITOCHONDRIAL FRACTIONS ISOLATED FROM PLANTS

Abstract— White light irradiation of a microsomal fraction from etiolated plants affects their ATP-dependent Ca²⁺ accumulation by inhibiting active uptake and enhancing passive efflux. The succinate-dependent mitochondrial Ca²⁺ accumulation as well is decreased by light. The wavelength dependence of these light effects as well as their low quantum yield suggest non-phototransformable protochloro-phyll(ide) [PChl(ide)] to be the photoreceptor. PChlide has been isolated from corn leaves pretreated with 5-amino levulinic acid. Addition of Pchlide causes photosensitization of an otherwise light insensitive microsomal Ca²⁺ accumulation. The observed light effect may be due to contamination of the mitochondrial as well as the microsomal fractions with PChl(ide)‡ containing particles. Irradiation of the intact tissue leads to the almost complete disappearance of the light effect on the in vitro Ca²⁺ accumulation.     

POLYNOIDIN: A MEMBRANE PHOTOPROTEIN ISOLATED FROM THE BIOLUMINESCENT SYSTEM OF SCALE-WORMS

Abstract— In order to isolate and purify the luminescence system of scale-worms, the scales were homogenized and extracted in the presence of Triton X-100. After chromatography on Bio-Gel A-5m, Sephadex G-75 and DEAE-cellulose, a single peak in luminescence activity was obtained. It showed properties of a membrane protein having a high mol. wt (about 500000) with characteristics of a photoprotein. The photoprotein, for which we suggest the name polynoidin , emits light in response to several reagents that can produce superoxide or hydroxyl radicals, such as H₂O₂ plus Fe²⁺, but the luminescence is not triggered by Ca^{2 +}. Oxygen is an absolute requirement for the luminescent reaction. The luminescence has a maximum at 510 nm. The photoprotein is not fluorescent when excited at 350 nm either before or after the luminescent reaction, thus differing distinctly from the green-fluorescent riboflavin in photosomes which is easily separated at the first step of the purification. We suggest a mechanism of the in vivo luminescence of scale worms in which the production of superoxide or hydroxyl radicals by the oxidation of reduced riboflavin could be regulated by Ca²⁺.     

CAFFEINE-ENHANCED PHOTOMOVEMENT IN THE CILIATE, STENTOR COERULEUS

The effects of caffeine, ionophores and calcium flux blockers on the step-up photophobic response, phototactic orientation and the intracellularly recorded, light-induced electrical action potential were studied in the ciliate, Stentor coeruleus . Caffeine alters the absorption and CD spectra and enhances the fluorescence of the photoreceptor pigment, stentorin. Independent of its effects on the spectroscopic properties of the photoreceptor pigment, caffeine shortens the photophobic response time by enhancing the Ca²⁺ conductivity of membranes, while Ca²⁺ flux blockers (LaCI₃ or ruthenium red) prolong it; both effects cancel each other. Evidence is presented that phototactic orientation is brought about by repetitive photophobic responses, since a change in the phobic response time results in a decreased accuracy of phototaxis.     

EFFECT OF PARAMAGNETIC SPECIES ON 8-METHOXYPSORALEN-SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE LAMBDA

Abstract. 8-Methoxypsoralen-sensitized photoinactivation of bacteriophage lambda (Λ_vir) was examined in the presence of various metallic ions. Paramagnetic ions, Co²⁺, Ni²⁺ or Mn²⁺, strongly repressed the reaction, whereas diamagnetic ions, Ca²⁺ or Zn²⁺, exerted no influence. Significant repression was also observed in the sample solution equilibrated with air.     

Contribution of the cAMP-Dependent Signal Pathway to Circadian Synchrony of Motility and Resting Membrane Potential in Paramecium

It is known that the ciliated protozoan Paramecium multimicronucleatum has synchronized circadian rhythms of motility, resting membrane potential and cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) concentrations. The present study shows that (1) extracellularly added 4 m M tetraethylammonium (TEA)⁺ (a K⁺ channel blocker) almost completely abolishes the diurnal oscillation of intracellular cAMP concentrations; (2) even 32 m^M TEA⁺ fails to abolish the circadian motility rhythm; but (3) the motility rhythm is highly damped when 4 m^M TEA⁺ and 100 μ^M CdCl₂ (a Ca²⁺ channel blocker) are added simultaneously. A cAMP analogue ( N ⁶-monobutyryl-cAMP) added extracellularly accelerates swimming velocity. Both a K⁺ channel blocker ( e.g . TEA⁺) and an inhibitor (trifluoperazine) of adenylate cyclase (AC) suppress cAMP formation, supporting the hypothesis that AC in Paramecium has dual functions, as a K⁺ channel and as an enzyme for cAMP formation. It is hypothesized that the circadian synchrony is due to circadian fluctuations of AC causing separate circadian changes both in ciliary motion and membrane potential through a cAMP-dependent signal pathway that forms a sophisticated network of second messengers to govern the synchrony together with Ca²⁺- and cGMP-dependent pathways in a manner antiphasic and/or complementary to one another.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.