ACTION SPECTRA FOR PHOTOACCUMULATION OF GREEN AND COLORLESS EUGLENA: EVIDENCE FOR IDENTIFICATION OF RECEPTOR PIGMENTS

Abstract —Phototaxis action spectra have been determined in three different types of Euglena gracilis ; green and dark-bleached cells containing the stigma and streptomycin-treated white mutant with an absorptionless stigma. In all three types of microorganisms the flavin containing paraflagellar body is present. The shape of the action spectrum is the same for the three types of cells and proportional to the absorption spectrum of flavoproteins. It has been shown that the structure of the action spectrum does not depend on the presence of screening organelles, on which, instead, depends the direction of the response to the light stimulus. It is concluded that the flavin chromophores present in the photoreceptor are the pigments responsible for phototaxis in Euglena gracilis.     

RED LIGHT-INDUCED ACCUMULATION OF EUGLENA GRACILIS

Abstract— It has been shown that green cells of Euglena gracilis accumulate under red light. The action spectrum of this response has been determined. Its shape and the results of regreening experiments clearly indicate a role of chlorophyll. The quenching effect of chlorophenyl–dimethylurea and NH₂OH demonstrate that this cell accumulation is not directly light-induced, as in the case of phototaxis, but is due to an effect of oxygen, the evolution of which is stimulated by light.     

PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF Euglena grcrcilis

Abstract —Phototaxis of the flagellate Euglena gracilis has been thought to be mediated by flavin photoreceptor molecules localized in the paraflagellar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-binding protein was solubilized and purified using nonionic detergents, high ionic strength, affinity Chromatography and standard column separations. Sodium dodecyl sulfate gel electrophoresis showed an apparent molecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K_D= 4 n M ) as well as under reducing conditions in the presence of dithionite (K_d= 6 n M ). Affinities towards flavin mononucleotide and flavin adenine dinucleotide were lower. Based on binding data and on estimates of the purified 68 kDa polypeptide, approximately lo⁶ flavin-binding sites were determined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.     

PHOTOPHOBIC BEHAVIORAL RESPONSES OF EUGLENA IN A LIGHT INTENSITY GRADIENT AND THE KINETICS OF PHOTORECEPTOR PIGMENT INTERCONVERSIONS*

Abstract— Accumulation of Euglena gracilis in small illuminated regions called light traps is due to a phobic response to the diminished light intensity at the boundary of the region. The rate of such accumulation of cells was measured as functions of both the light intensity within the trap and the change of intensity at the boundary of the trap. The initial rate of accumulation of a population of cells was taken to be a direct measure of the phobic response of a single typical cell. The data indicate that the strength of the behavioral response in a single cell may be described as being proportional, to the rate of change of the amount of photochemically active form of a photoreceptor pigment molecule which can exist in two predominant forms.     

FOURIER ANALYSIS OF ANGULAR DISTRIBUTIONS FOR MOTILE MICROORGANISMS

The method of Fourier transformation is used to analyze histograms of the angular distribution of organisms moving with respect to a stimulus, in this case the light direction. In the Fourier spectra, the components with the highest amplitudes indicate the modality of orientation, i.e. the number of preferred directions, and the corresponding phase values indicate the deviation from the light direction. In the frequency domain, those components with high amplitude and/or those with low frequencies (low-pass filtering) can be selected and used to reconstruct a smoothed histogram by means of an inverse Fourier transform. This method reduces the noise level and reveals the prominent features of histograms. Examples of unimodal, bimodal and multimodal distributions are shown in their original and smoothed form for the flagellate Euglena gracilis , and for amoebae and pseudoplasmodia or the slime mold Dictyostelium discoideum.     

FLUORESCENCE OF Euglena gracilis PHOTORECEPTOR PIGMENT: AN in vivo MICROSPECTROFLUOROMETRIC STUDY

Abstract— The spectroscopic characterization of the photoreceptor pigment is one of the main questions in the study of the photosensory transduction chains in photomotile microorganisms. One of the possible techniques that can be used is in vivo microspectrofluorometry. By means of a tunable dye-laser microspectrofiuorometer developed by us, we have investigated some of the spectroscopic properties of the photoreceptor pigment of the green flagellate Euglena gracilis. The in vivo fluorescence excitation spectrum has been determined and the fluorescence quantum yield has been measured. The results show that flavins are indeed present in the paraflagellar body of E. gracilis and that their fluorescence quantum yield is much lower than that of a free flavin. An estimate of the order of magnitude of the rate constants for primary molecular reactions is tentatively given.     

Zur Optimierung der Bestrahlungsbedingungen im Bremsstrahlfeld des Mikrotrons MT 22 des vik Dubna

In the bremsstrahlung field of the Microtron MT 22, copper foils (0.035 mm thick) positioned along beam direction and also perpendicular to the beam at different distances from the bremsstrahlung target were irradiated. From these activated copper foils autorodiograms were prepared. By means of a densitron and a microdensitometer, the autoradiograms were measured. The densitron pictures give a qualitative impression of the geometry of the bremsstrahlung field, whereas the curves of optical density yield quantitative values. From optical density curves, conclusions have been drawn about optimal geometry for irradiation positions of samples in this bremsstrahlung field. A sample rotating device applicable for photon activation analysis by means of the MT 22 is described.      

REAL TIME COMPUTER-CONTROLLED TRACKING OF MOTILE MICROORGANISMS

Abstract— A hardware and software system is described which is capable of automatically following the movement of microorganisms and analyzing the deviation of individuals in a population from a predefined direction. The image of the moving organisms is viewed by a video camera, digitized in real time and stored in a dedicated video memory holding 512 times 256 pixels with 64 grey levels. A microcomputer has access to this video memory via four parallel ports. The software analyzes the position of an organism selected randomly and attempts to follow its track for a predefined period of time. If successful, it calculates the direction of movement and stores the data in a file for statistical analysis.
Using this system, the movement of populations of the unicellular flagellate, Euglena gracilis, in unilateral and bilateral light beams has been studied and compared with the dark behavior. When irradiated from two opposite light sources of equal fluence rates half of the population swims towards either light source. However, when the light sources are oriented at an angle of 90° to each other the cells move on the resultant away from the light. These results confirm the shading hypothesis for negative phototaxis.     

LIGHT-INDUCED ACCUMULATIONS OF DICTYOSTELIUM DISCOIDEUM AMOEBAE

Abstract— Light-induced accumulations of amoebae of Dictyostelium discoideum in 'light traps' have been observed. The greatest accumulations are obtained with cell densities of about 8 × 10⁴ cells/mm². Accumulations are observed at incident fluence rates over about one decade both for white and for monochromatic light; higher fluence rates cause dispersal from the 'light trap'. An action spectrum for the photoaccumulation, calculated from fluence-response curves using the zero thresholds, shows a major peak between 405 and 410 nm and extends through most of the visible spectrum. This action spectrum does not coincide with any of the pigments known to be present in D. discoideum . The cellular basis for the photoaccumulation has been studied. No light effects on cell divisions or cell aggregation are observed during the 2 h duration of an accumulation experiment. Microvideographic analysis of single amoebae is consistent with the hypothesis that the amoebae are positively phototactic and move toward the light scattered from cells in the 'light trap', thus accumulating in the trap.     

BIPHASIC ENERGY CONVERSION KINETICS AND ABSORBANCE DIFFERENCE SPECTRA OF PHOTOSYSTEM II OF CHLOROPLASTS. EVIDENCE FOR TWO DIFFERENT PHOTOSYSTEM II REACTION CENTERS

Abstract— The continuous illumination induced kinetics of photochemical energy conversion at system II have been measured with isolated and 3-(3, 4-dichlorophenyl)-l, l-dimethylurea (DCMU) poisoned chloroplasts by means of absorbance difference spectroscopy in the UV and by the area growth over the fluorescence induction curve at room temperature. An optimal set of conditions was found in order to isolate absorbance changes caused by the reduction of the primary electron acceptor Q of PS II by suppressing other electron transfer processes. The light induced kinetics of Q- accumulation in the absorbance change measurements were found to be biphasic and strictly correlated with the kinetics of the area growth measured under the same conditions. From the resolution of the biphasic kinetics at different wavelengths in the UV region of the spectrum, it was found that both kinetic components in the system II photochemistry involve the reduction of a plastoquinone molecule to its plastosemiquinone anion. From the two kinetic components one was fast and non-exponential and the other relatively slow with an exponential time course. The initial rate difference in the kinetics of the two components was by a factor of approximately 3. A difference by a factor of about three was also found in the flash saturation curves of the two kinetic components.
The results are explained by the hypothesis that in higher plant chloroplasts there are system II reaction centers embedded in a large pigment matrix with statistical energy transfer, and system II reaction centers embedded in separate, in terms of excitation energy transfer, units. The effective absorption cross section per reaction center for the centers in the statistical pigment bed is approximately 3 times larger than that of the reaction centers in the separate system II units. The two types of system II reaction centers have different yields of excitation trapping and charge stabilization properties.     

FLUOROMETRIC EVIDENCE FOR FLAVINS IN ISOLATED EYESPOTS OF EUGLENA GRACILIS VAR. BACILLARIS

Abstract— Fluorometric evidence suggesting the presence of flavins in isolated eyespots of Euglena gracilis var. bacillaris is reported for the first time. Fluorescence spectra of eyespots and flavin standards show maxima at 540nm and 530nm, respectively. Excitation spectra show matching major peaks at 360–370 nm and at 450nm. The addition of riboflavin standard to eyespot samples increases fluorescence intensity without major corresponding shifts in wavelength maxima. Photolysis of eyespot samples in the presence of EDTA effects a decrease in the fluorescence intensity; the fluorescence is quantitatively restored to its initial value by bubbling the photolyzed solution with air. Preliminary quantitative data, obtained by fluorescence measurements, indicate the presence of ca. 5 × 10^-4μg flavin/ml eyespot sample. While flavins have been hypothesized to be components of the photoreceptor system, they have been reported previously only in the paraflagellar bodies of intact cells. Emission and excitation data obtained by us for eyespots are similar to those previously reported by other investigators for paraflagellar bodies, but our studies now suggest the presence of flavins also in Euglena eyespots.     

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.     

THE EFFECT OF POTASSIUM IODIDE ON PHOTOPHOBIC RESPONSES IN EUGLENA: EVIDENCE FOR TWO PHOTORECEPTOR PIGMENTS*

Abstract— Potassium iodide, a quencher of flavin fluorescence, inhibits the shock reaction which Euglena experiences upon a sudden decrease in light intensity (inverse photophobic response) completely at a concentration of 150 mM. The rate of swimming of the cells at the same concentration of KI is reduced to 30% of the control. The direct photophobic response, a shock reaction which appears identical but occurs upon an increase in light intensity, is unaffected by KI as is negative phototaxis of Euglena. It is concluded that a non-flavin pigment system mediates photoreception for the direct photophobic response and negative phototaxis.     

Photocontrol of organelle and cell type specific changes in the polypeptide composition of Euglena and sorghum

Two-dimensional (2-D) gel electrophoresis has been used to follow changes in cell type specific and organelle localized polypeptides upon exposure of etiolated sorghum shoots and dark-grown resting Euglena to light. Total protein extracted from isolated bundle sheath strands and mesophyll protoplasts was resolved by 2-D gel electrophoresis. The cell type specific polypeptides were localized on the whole shoot 2-D gel map in order to determine changes in the levels of these polypeptides upon light exposure. An image analyzer was used to analyze fluorographs of 2-D gels of total Euglena protein pulse-labeled with [35S]sulfate in the dark, immediately upon light exposure and 1, 4, 14, 24, 48 and 72 h after light exposure. The subset of polypeptides whose relative rate of synthesis changes more than threefold immediately upon light exposure was identified. The different patterns of changes in the rate of synthesis of this subset of polypeptides were followed.     

Determination of two metals from a single potentiometric titration curve The application of two indicator electrodes

The advantages of applying two indicator electrodes in complexometric potentiometric multicomponent titration are shown by means of simulated titration curves. Two measurement arrangements have been considered, one in which the indicator electrodes are directly connected to a voltmeter and the other in which the electrodes are connected to the voltmeter through a summing operational amplifier. They have been compared with the conventional arrangement of a single indicator electrode and a reference electrode. The influence of the stability constants of the complexes in solution and of the electrode parameters on the shape of titration curves has been examined. It is shown that the use of two indicator electrodes may significantly increase the applicability of multicomponent potentiometric titrations.     

顺式1,4-聚丁二烯的流变性质——Ⅰ.流动曲线的不连续性

用毛细管流变仪测定了四种不同催化体系高顺式1,4-聚丁二烯的流动曲线。流动曲线由连续变成不连续的临界温度随顺式含量的增加而增高。并发现长链支化也起重要作用,线性程度愈好则临界温度也愈高。挤出物的不同形状可作如下描述:流动不稳定性可独立地发生于口模入口、口模出口及模壁,但滑移本身并不导致外形不规整。     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

Complex electrooptic research of nano-particle parameters in colloids

A research of some colloids has been carried out by means of dynamic light scattering, electrooptical and magnetooptical techniques. Intensity autocorrelation functions of scattered light have been compared to the relaxation curves of electrooptical effect for colloid particles of different shapes. The results of complex research confirm that the complicated character of light scattering by particles allows us to use the methods of birefringence and dichroism only formally when studying most colloid systems. Very thin nano-disperse structures are an exception to this rule. The investigation of polydispersity of some colloids was carried out by magnetooptical and two electrooptical techniques. Size distribution functions resulted from the different techniques agree. This justifies the suppositions about particle light scattering that are required for the use of the methods.     

DOSE RESPONSE BEHAVIOUR FOR POLAROTROPISM OF THE CHLORONEMA OF THE FERN DRYOPTERIS FILIX-MAS (L.) SCHOTT

Abstract— The dose response behaviour for polarotropism of the chloronema of the fern Dryopteris filix-mas was studied in detail. By varying the two factors, time and intensity, dose response curves were obtained, which consisted of as many as two maxima and minima or no maxima and minima at all. Furthermore, the effects of red light and dark, given before polarotropic induction, were compared, and the influence of temperature on the dose response behaviour was examined. There are two main results: (1) Under all conditions tested, the dose response behaviour in far-red, red, blue, and near U.V. apparently is the same. (2) In this one system, the chloronema of Dryopteris , all types of dose response curves, which have been described previously for tropic responses mediated by light, could be obtained simply by varying the conditions. The variable and complex dose response behaviour is discussed in connection with other photoresponses in lower and higher plants.     

ULTRAVIOLET-INDUCED APOCHLOROSIS AND PHOTOREACTIVATION IN TWO STRAINS OF EUGLENA GRACILIS*

Abstract— Very low doses of ultraviolet irradiation result in a complete loss of the ability to synthesize chlorophyll in two closely related strains of Euglena gracilis (var. hacillaris and strain Z). Both strains are equally sensitive in this response under non-photoreactivating conditions. The ability to synthesize chlorophyll is completely photoreactivated by visible light in E. grncilis var. bacillaris , even after lethal doses of u.v.: the Z strain. however, while photoreactivable following low doses of u.v., remains bleached after doses adequate to kill only 10–20 per cent of the cells.