PHOTORECEPTOR PIGMENT IN Blepharisma: H+ RELEASE FROM RED PIGMENT

In faded cells of Blepharisma kept in a standard saline solution containing bacteria which had been cultured on agar plates containing glucose and polypepton, threshold light intensity for step-up photophobic response elevated. This result suggests that red pigment (blepharismin) contained in Blepharisma cells is involved in the step-up photophobic response. The pH of the aqueous solution of the red pigment was found to decrease when light was applied, indicating that the pigment releases H⁺ in response to light stimulation. However, faded pigment preparation by light irradiation did not show pH decrease. In the living cells faded by light irradiation, threshold light intensity for the step-up photophobic response was raised. Results suggest that H⁺ release from the red pigment induced by light irradiation might be responsible for the step-up photophobic response of the cells.     

Front Cover: Tetrathiaporphyrine: A Novel Natural Product from Allium Subgenus Melanocrommyum (Eur. J. Org. Chem. 20/2023)

A novel purple-red pigment from the plant genus of Allium (onions, leeks), subgenus Melanocrommyum (drumstick onions), is described. Because of this pigment, which reminds of blood, some species of the subgenus Melanocrommyum are used in Southwest and Central Asia as herbal medications, as spices or as vegetables. A proposal for the formation of this structure is provided, which is the first example of a tetrathiaporphyrin in nature. The described substance shows low stability towards thermal stress, light and oxidant; additionally, its symmetric nature made structural elucidation a challenge. It could be shown that, contrary to previous reports, the structure of the red pigment is that of a tetrathiaporphyrin. Full structure elucidation was only possible by X-ray crystallography and total synthesis of the intended molecule. Further on, the newly discovered red pigment was used for the modification of nanoparticles and monitoring its polymerization events using SPR.     

Pteridines. Part C. Structure and nonenzymatic synthesis of aurodrosopterin

The nonenzymatic synthesis of aurodrosopterin ( 5 ) from 6-acetyl-2-amino-3, 7, 8, 9-tetrahydro-4H-pyrimido-[4,5-b][1,4]diazepin-4-one ( 3 ) and 7,8-dihydrolumazine ( 4 ) at pH 3 (HCl) was performed. The identity of the synthesized compound with the natural eye pigment isolated from drosophila heads was confirmed by thin-layer chromatography on cellulose and by comparisons of the ¹H-NMR and UV/VIS spectra. The nonenzymatic synthesis of a neodrosopterin-like red pigment from 3 and 2,4-diamino-7,8-dihydropteridine was also carried out, but its identity could not be established. This pigment, called aminodrosopterin, has an absorption peak at 489 nm, which is very close to that of neodrosopterin.     

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.     

Toxic and Phototoxic Properties of the Protozoan Pigments Blepharismin and Oxyblepharismin*

Abstract— The toxic and phototoxic properties of blepharismin and oxyblepharismin that were purified from the pigments of a ciliated protozoan, Blepharisma japonicum, by thin-layer chromatography, were investigated in detail. The toxicity was tested against the ciliated protozoan Dileptus margaritifer, which is relatively sensitive to blepharismin. Although oxyblepharismin has been believed to be neither toxic nor phototoxic, it was found that oxyblepharismin is toxic in the dark and is also phototoxic. This shows that oxyblepharismin can act as a photosensitizer. The toxicity and phototoxicity of these pigments were compared with those of hypericin, which is known to be a typical, strong photosensitizer from plants. It was concluded that blepharismin and oxyblepharismin have strong intrinsic toxicities in the dark compared with hypericin, but their phototoxicities are slightly weaker than that of hypericin. This strong intrinsic toxicity supports our proposal that blepharismin acts as a defensive device against predators in the dark as well as in the light. The decrease in the defensive ability and the increase in the resistance to photokilling of Blepharisma concomitant with its color change from red to blue-purple in response to weak illumination can be explained by the decrease in toxicity and phototoxicity of the pigment itself and by the decrease in amount of the pigment.     

7-cis-porphyropsin from 7-cis-3-dehydroretinal and cattle opsin.

Abstract 7-cis-3-Dehydroretinol, prepared by photoisomerization of the all-trans isomer in a polar solvent, was found to combine with cattle opsin to form a visual pigment analogue with an absorption maximum at 464 nm. The pigment is only moderately stable in hydroxylamine or in Ammonyx LO. and cannot be purified by column chromatography using Ammonyx LO. 9-cis-Porphyropsin, prepared in a manner similar to that reported by Azuma et al., is stable and has been purified by passing through a hydroxylapatite column. Three fractions were collected with eluant of increasing ionic strength. All fractions exhibit similar absorption properties with Λ_max at 498 nm. These results further indicate that the binding of opsin is a flexible one.     

Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata

The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm⁻² UVA, 0.07 and 0.9 J cm⁻² UVB, 20 n m β-PDH (pigment dispersing hormone) or 10 μ m SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo . Cultured cells were exposed to 250 μ m L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo . SIN-1 did not induce pigment dispersion in the cell cultures. l-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.     

The Protective Effect of Violaxanthin from Nannochloropsis oceanica against Ultraviolet B‐Induced Damage in Normal Human Dermal Fibroblasts

Skin photoaging, which is mainly induced by ultraviolet B (UVB) radiation, is prevented by the application of UV‐protective agents. The microalga Nannochloropsis oceanica (N. oceanica) has been primarily reported as a potential biofuel; however, in this study, we investigated whether N. oceanica extracts exerted photoprotective effects against UVB‐irradiated human dermal fibroblasts (HDFs) and which single component was responsible for the protective effect of the extracts. Two extracts—pigment and nonpigment—were prepared from N. oceanica biomass. WST‐1 assay and expression analysis of interleukin genes showed that the pigment extracts were not significantly cytotoxic to HDFs. Further experiments revealed that treatment with the pigment extract upregulated the expression of collagen genes and significantly blocked UVB‐induced damage such as decreased cell viability and increased ROS production. Next, to investigate the pigment composition of the extracts, HPLC analysis was conducted and violaxanthin was identified as the major pigment. The UVB photoprotective effect of the pigment extracts was confirmed in violaxanthin‐treated HDFs. In addition, violaxanthin significantly attenuated UVB‐induced G1 phase arrest, senescence‐associated β‐galactosidase activation, p16 and p21 upregulation, ERK phosphorylation and the downregulation of ECM molecules in HDFs. Therefore, we concluded that violaxanthin was a potential antiphotoaging agent.     

Localization of Blepharismin Photosensors and Identification of a Photoreceptor Complex Mediating the Step-up Photophobic Response of the Unicellular Organism, Blepharisma

Abstract— Photosensitivity for the step-up photophobic response of Blepharisma is localized in the anterior 1/5 of the cell body. Blepharismin pigment, which is believed to be a photoreceptor pigment mediating the step-up photophobic response of the cells, was separated into five types of blepharismin (BL-1, -2, -3, -4 and -5). Blepharismin-1, -3, -4 and -5 were localized in the posterior 4/5, while BL-2 was located over the entire cell body; the anterior end, which is the photosensitive region, contained only BL-2. The results indicate that a functional photoreceptor pigment mediating the step-up photophobic response is BL-2. Hydroxylapatite chromatography revealed that BL-2 was bound to a 200 kDa membrane protein. We concluded that a photoreceptor mediating the step-up photophobic response was a BL-2/200 kDa protein complex.     

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.     

STUDIES ON THE RED AND BLUE FORMS OF THE PIGMENT OF BLEPHARISMA*

Abstract— –The intracellular pigment of the ciliate protozoan Blepharisma in the presence of oxygen sensitizes the cells to bright visible light (2700 foot candles (fc)). Illumination of the cells with dim visible light (200 fc) changes the color of the pigment to blue-gray; such cells are no longer sensitive to bright visible light. The pigment which exists in granules can be extruded by cold treatment and is slowly regenerated. The suspension of red cells, the extruded pigment from them and an ethanol extract of the pigment all have very similar absorption spectra. Illumination of the red pigment in ethanol converts it to the blue form of the pigment but only if oxygen is present, indicating a photooxidation. The pigment can be oxidized in darkness to the blue form by ozonation. A suspension of blue cells, the extruded pigment from them and an ethanol extract from them, all have similar absorption spectra. The pigments in red and blue form are very similar spectrophotometrically and in solubility in three species of Blepharisma studies: B. americanum, B. intermedium and B. japonicum. The purified pigment has strong absorption in the far (200–300 nm) ultraviolet (u.v.) and may serve as a screen against damaging U.V. radiation, especially as Blepharisma shows poor photoreactivation.     

INTRACELLULAR DICHROIC ORIENTATION OF THE BLUE LIGHT-ABSORBING PIGMENT AND THE BLUE-ABSORPTION BAND OF THE RED-ABSORBING FORM OF PHYTOCHROME RESPONSIBLE FOR PHOTOTROPISM OF THE FERN Adiantum PROTONEMATA

The intracellular localization and orientation of the receptors for the blue light-induced phototropism in the fern Adianrum protonemata, phytochrome and the blue light-absorbing pigment, were investigated by combining the techniques of cell centrifugation and of microbeam irradiation with linearly polarized light. The phototropic response was induced in the cells even after they had been centrifuged basipetally to spin down the endoplasm from the apical region. When a polarized blue microbeam was given to a flank of the apical region of the protonema, the phototropic response after compensation of phytochrome effect by far-red light was most effectively induced when the polarization plane was parallel to the long axis of the cell. If protonemata were pre-irradiated with blue and far-red light, the phototropic response was mediated through phytochrome alone. If such pre-irradiated protonemata were similarly irradiated with a polarized blue microbeam, polarized light vibrating parallel to the cell axis was again most effective in inducing the response. These results indicate that both the blue light-absorbing pigment and the phytochrome responsible for the blue light-induced phototropism in Adiantum are confined to the plasma membrane and/or the ectoplasm and that the transition moments of their blue-absorption bands are nearly parallel to the cell surface.     

Extraction and partial characterisation of antioxidant pigment produced by Chryseobacterium sp. kr6

Pigments synthesised by Chryseobacterium sp. kr6 growing on feather waste were extracted and characterised. The pigment extract was characterised by KOH test, UV–vis, CIELAB colour system, HPLC-DAD-MS, FTIR and its antioxidant capacity was evaluated. A positive bathochromic shift was observed when kr6 colonies or pigment extracts were subjected to alkaline solution (20% KOH) and a λ_max at 450 nm was detected for acetone extracts, although no typical fine structure of carotenoids was detected in the electomagnetic spectra. The HPLC profile of the extracted pigment showed that the compound has three different peaks with λ_max near 450 nm. The FTIR analysis shows some principal functional groups from a flexirubin-like molecule. The pigmented compound also presents antioxidant activity evaluated by the scavenging of the ABTS radical.     

Carbazoledioxazines having long alkyl groups. 3. Syntheses and properties of halogen free carbazoledioxazines

Long chain derivatives of halogen free carbazoledioxazine which is a typical organic pigment with a linear type structure (5,15-dialkyl-5,15-dihydrodiindolo[3,2-b:3′,2′-m]triphenodioxazines) and an angular type structure (5,15-dialkyl-7,17-dialkoxy-5,15-dihydrodiindolo[2,3-c:2′,3′-n]triphenodioxazines) were newly synthesized. Their structures were confirmed by ¹H-nmr, ms and elemental analysis. The thermal, spectroscopical and electrochemical properties were investigated by means of DSC, uv-vis and cyclic voltammetry.     

Energy transfer between photosynthetic units analyzed by flash oxygen yield vs. flash intensity

Abstract— Relative yield of O₂ (Y) was measured in Chlorella pyrenoidosa in response to varied intensity (l) of single 10μsec flashes on a constant low background of 710 nm light. Analysis is based on the proposition that the photochemical event leading to O₂ evolution occurs at a reaction center or trap which requires a time much longer than the flash for regeneration by dark reactions. Hence O₂, flash yield measures the number of traps ‘killed’ and allows treatment in terms of target theory. Data for Y vs. l were analyzed by computer fitting to four models. The first three models supposed that each unit (aggregate of light-harvesting pigment molecules) contains one, two, and three traps, respectively, and allows no transfer of excitation energy out of the unit. The last model supposed only one trap per unit and a probability of transfer out of a unit with closed trap. Among the first three models, the data best fit the one with two traps per unit. A slightly better fit for two traps per unit was obtained by introducing a trapping efficiency less than unity. An equally good fit was also obtained with the model of the Joliots with a probability of 0.3 that excitation energy in a unit with closed trap is transferred to another unit. Uncertainties in analysis arose from the necessity of treating maximum flash yield as an estimated parameter and by the possible inhomogeneity in units and traps.     

STUDIES ON PHOTOTROPISM OF YOUNG SPORANGIOPHORES OF PILOBOLUS KLEINII*

Abstract— Young sporangiophores of the fungus, Pilobolus kleinii, respond to unilateral illumination by bending or by growing toward light of wavelengths between 312 and 530 mμ, with peaks of sensitivity near 360 and 450 mμ. Young sporangiophores exhibit a negative phototropic response to wavelengths shorter than 300 mμ, with a strong negative response at 280 mμ. Since the action spectrum did not correspond to the absorption spectrum of the pigmented zone as measured in vivo, and since colorless sporangiophores formed on media containing diphenylamine were capable of phototropic response, it is unlikely that the conspicuous orange-yellow pigment in young sporangiophores is the photoreceptor for phototropism. The results of probing with small beams of light and the behavior of sporangiophores submerged in mineral oil, together with measurements of the refractive index of the tip and base indicate that the photosensitive region is located in the tip of the young sporangiophore.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

STEADY HYPSOCHROMIC SHIFT OF PEAK RESPONSIVITY TO ATTRACTANT LIGHT FROM 590 nm TO 560 nm IN Halobacterium halobium*

Abstract— Peak responsivity of photoattraction in Halobacterium halobium cells shows steady hypsochromic shift from 590 nm wavelength under low irradiance conditions to 560 nm under high irradiance conditions. Inversion of the photoattractant response, as dependent on blue vs red background light, is compatible with the known properties of photochromic sensory rhodopsin-I (SR-I) with ground state maximum absorption at 587 nm. Relaxation of the photoattractant response in H. halobium, as a function of wavelength and irradiance, gives a hint at an antagonistic pigment or intermediate state, different from ground state SR-I, with peak sensitivity at 620 nm or even above. The less sensitive photoattractant response at 560 nm persists without photorelaxation and represents the peak responsivity under high irradiance conditions.     

Distinguishing Activities in the Photodynamic Arsenals of the Pigmented Ciliates Blepharisma sinuosum Sawaya, 1940 and Blepharisma japonicum Suzuki, 1954 (Ciliophora: Heterotrichea)

Blepharismins are photodynamic hypericin-like dianthrones produced as a variable pigment blend in Blepharisma ciliates and mostly studied in the Afro-Asiatic Blepharisma japonicum. The present work describes the bioactivity of pigments from the Brazilian Blepharisma sinuosum. Comparative analyses showed that the pigments from both species can trigger photo-induced modifications in phospholipids, but different redox properties and biological activities were assigned for each pigment blend. Stronger activities were detected for B. sinuosum pigments, with the lethal concentration LC₅₀ 10 × lower than B. japonicum pigments in light-irradiated tests against Bacillus cereus and less than half for treatments on the human HeLa tumor cells. HPLC showed B. sinuosum producing a simpler pigment blend, mostly with the blepharismin-C (~ 70%) and blepharismin-E (~ 30%) types. Each blepharismin engaged a specific dose–response profile on sensitive cells. The blepharismin-B and blepharismin-C were the most toxic pigments, showing LC₅₀ ~ 2.5–3.0 µm and ~ 100 µm on B. cereus and HeLa cells, respectively, after illumination. Similarity clustering analysis compiling the bioactivity data revealed two groups of blepharismins: the most active, B and C, and the less active, A, D and E. The B. sinuosum pigment blend includes one representative of each clade. Functional and medical implications are discussed.     

CHLOROPHYLL CATABOLISM. PART 3. STRUCTURE ELUCIDATION AND PARTIAL SYNTHESIS OF A NEW RED BILIN DERIVATIVE FROM Chlorella protothecoides*

The structure of a new red pigment produced during the “bleaching” of Chlorella protorhecoides cells has been elucidated by analytical methods as well as by in vitro isomerization into the new compound of a synthetic sample of a previously characteriLed chlorophyll a catabolite.