Photooxidations in pigmented Blepharisma

1. Blepharisma undulans, a protozoan with a reddish pigment, shows increased oxygen consumption under the influence of light. 2. If the light intensity is high, the animals are killed during a burst of oxygen consumption. 3. If the blepharismas are first bleached by exposure to light of low intensity they show only slightly increased oxygen consumption under the influence of light and they are not killed. 4. A preparation in which the animals are killed by heat still shows the increase in oxygen consumption on illumination with brilliant light. The supernatant solution does so as well, as does an alcohol extract of the dye. 5. The conclusion is drawn that the blepharismas are killed during photooxidation of the pigment, but the mechanism of action is not clear. Several possibilities are considered in the discussion.     

Action Spectra for Step-Up Photophobic Response in Blepharisma

ABSTRACT The cells of Blepharisma which possess red pigment (blepharismin) show step-up photophobic response (temporal ciliary reversal induced by a sudden increase in light intensity). Bleaching of the cells by cold shock raised a threshold light intensity for the response, Oxidation of red pigment that produced blue pigment did not raise the threshold for the response. The action spectrum for the step-up photophobic response of the cells which possess normal red pigment had peaks at about 580, 540 and 490 nm, a value which coincided with peaks of an absorption spectrum of the red pigment. The absorption spectrum of oxidized pigment (blue pigment) shifted 20 nm toward infrared light. The action spectrum for the response of the cells which possess blue pigment also shifted 20 nm toward infrared light. Results suggest that red pigment might be involved in the step-up photophobic response. Key words. Blepharismin, ciliary reversal, photoreceptors, photoresponse.     

Low Temperature Absorption Spectra of Chlorophyll a in Polar and Nonpolar Solvents

Absorption spectra of chlorophyll a were measured in polar and non-polar solvents, as a function of temperature from 298 degrees to 77 degrees K. Both dilute and concentrated solutions were examined. In both types of solvents at room temperature, the absorption spectra of concentrated solutions differ from dilute ones in that the half width of the main red absorption band is greater, and all bands are shifted to longer wavelengths. These differences are largely due to the presence of dimers when the pigment concentration is high. In dilute ethanol solutions, where the chlorophyll is unassociated, cooling causes a red shift in all bands which is due to the increased polarity of the solvent at low temperature. On cooling at high concentrations in ethanol and EPA, a new band appears near 700 nm. This band is attributed to dimers present prior to cooling, but absorbing at shorter wavelengths at room temperature. In nonpolar solvents, a band near 700 nm appears at the solvent freezing point. In these solvents, the "700" nm absorption is attributed to dimers, and/or small polymers, partly formed by cooling. A change in aggregate geometry when the solvent becomes viscous or frozen can account for the appearance of this "700" nm absorption band at low temperature, in polar and nonpolar media.     

真眼点藻类色素的提取与测定方法

分别采用甲醇、乙醇和丙酮3种有机溶剂提取7种真眼点藻的色素,比较3种有机溶剂提取色素的效果,测定3种有机溶剂色素提取液的吸收光谱,利用分光光度法计算藻的叶绿素a和类胡萝卜素的含量,并比较甲醇和乙醇色素提取液在A₄₇₀和A₆₆₆的最大吸收峰。结果表明:使用乙醇比甲醇和90%丙酮操作更简便、快捷并且毒害低。3种有机溶剂色素提取液的叶绿素a和类胡萝卜素的含量均无显著性差异(P>0.05),提取率基本一致。色素在3种有机溶剂中的吸收光谱相似,甲醇和乙醇的色素提取液在A₄₇₀和A₆₆₆的最大吸收峰并无显著性差异(P>0.05)。乙醇色素提取液可使用Lichtenthaler的公式计算色素含量。     

不同北虫草菌株产类胡萝卜素分析

通过对不同来源北虫草菌株子实体中提取的色素进行定性分析并对其含量进行测定,获得类胡萝卜素含量高的北虫草菌株。结果表明,浓硫酸反应中溶液在两相交界处呈现特征性的蓝绿色,色素提取液在400～600 nm范围内有波长分别为415、440和460 nm的3个吸收峰与类胡萝卜素标准的吸收光谱一致,证明子实体中提取的色素为类胡萝卜素;6个菌株子实体类胡萝卜素的含量差异较大,且J菌株的类胡萝卜素的含量最高,达到5.021 mg/g。     

中国土壤原生动物新纪录种(纤毛虫门：多膜纲；异毛目)

报道了首次在中国土壤中鉴定到的、隶属于纤毛虫门多膜纲异毛目的７种原生动物。这些种类是泥炭藓苔扭虫、透明赭虫、长赭虫、透明扭头虫、弯曲扭头虫、旋短端突虫和梅氏扁豆虫。     

理化因子导致梅花'南京红'花色色素的颜色变化

梅花是中国的候选国花之一。属于花色苷的梅花'南京红'花色色素用含1％浓盐酸(v／v)的甲醇提取,并呈现纯净的紫红色。体外试验表明:该色素在pH0-3范围内颜色稳定,因不同光质、热、氧化剂、还原剂、螯合剂而呈现无色、墨绿色或黄绿色,因不同金属离子、离子的不同浓度而呈现程度不同的红色、紫色、黑黄色、红中带黑或微蓝绿色,葡萄糖和低浓度苯甲酸钠几乎不影响其色泽,蔗糖使颜色变淡,柠檬酸却使其颜色变深。该文可为梅花红色花色的机理探索、梅花花色苷的分子结构鉴定、梅花红色花色色素的开发利用提供参考和前提。     

Pigment formation in the dermatophytes

Avstract The moist mycelium ofT. tonsurans was extracted with 3 % w/v sodium hydroxide to yield a purple to violet solution which was passed through a cation-exchange resin column, evaporated partially, dialyzed and lyophilized. The lyophilized powder was extracted using absolute ethanol, acetone and chloroform (Process A) and separately by defatting with petroleum ether and n-hexane followed by chloroform-methanol 2:1 (Process B).Separation was achieved for the extract of Process B on thin layer chromatography using Silica Gel G and butanol-ethanol-water as the solvent system. Ultra-violet and visible spectrophotometric absorption data are reported for the lyophilized extract ofT. tonsurans, hydrolyzates of the lyophilized extract, and for sodium hydroxide extracts ofT. megnini, T. mentagrophytes, T. rubrum, T. tonsurans andT. violaceum. The spectra were similar in all cases.A review of pigment substance in dermatophytes is made, which together with the experimental data secured withT. tonsurans and previous experimentation, suggest the possible effects of drying procedures, contusion, solvents, acidic additives and method of extraction on the removal of pigment from the mycelium of dermatophytes. It is suggested that all the above conditions influence the substances ultimately extracted and that some alteration of a basic pigment association in the organism occurs to release pigment fragments or fractions of varying complexity. Oxidation and polymerization may influence the end result. It is possible that the mechanism presented can account for the number of pigment substances reportedly occurring in the dermatophytes and that a similar pigment structure or complex exists in other Trichophyton species.Grateful thanks are extended to the Medical Research Council of Canada for financial support.     

Further evidence that the pigment in the Dubin-Johnson syndrome is not melanin

The pigment in the Dubin-Johnson syndrome (DJS) is shown unequivocally not to be a typical melanin or closely related polymer. In electron spin resonance (ESR) studies of DJS pigment from a hepatoma, it is shown that, unlike true melanins, the pigment associated with the DJS syndrome has no free radical in the absence of light. Exposure to even low levels of visible light over a broad frequency range induces a free radical in the DJS pigment. Previous studies did not appreciate the sensitivity to light of this pigment and therefore erroneously concluded that the DJS pigment had a permanent free radical. The light induced ESR signal in DJS tissue has spectroscopic properties that differ significantly from any known melanins. The pigment is not extracted by lipophilic solvents and is centrifuged down at 50,000g, but not at 5,000g.     

Ommochrome pigments in the eyes of Euphausia superba (Crustacea,Euphausiacea)

Summary Ommin and a xanthommatin-like ommochrome pigment have been extracted from the eyes of Euphausia superba, and identified by their absorbance spectra and redox reactions invarious solvents. The absorbance spectrum of ommin is relatively stable, but that of the xanthommatin pigment is sensitive to light, pH, and hydroxylamine. Both pigments are readily soluble in 2% digitonin. They are therefore likely contaminants of visual pigment extracts, and may consequently introduce artifacts into visual pigment characterizations. Microspectrophotometric measurements show that both pigments are components of the distal and proximal screening pigment granules in E. superba ommatidia.This paper is dedicated to the memory of Professor Mary Alice McWhinnie, to whom I owe a great debt of gratitude for her patient tutelage and the opportunity to participate in her Antarctic research, for the encouragement to pursue my own interests, and for her generous friendship. It was a cherished association which ended sadly with her untimely death in March, 1980. She will always be fondly remembered     

THE EFFECTIVENESS OF THE SPECTRUM IN CHLOROPHYLL FORMATION

1. Although the carotenoid pigments are present in large concentration in the plastids of etiolated Avena seedlings as compared with protochlorophyll, the pigment precursor of chlorophyll, it is possible to show that the carotenoids do not act as filters of the light incident on the plant in the blue region of the spectrum where they absorb heavily. This suggests that the carotenoids are located behind the protochlorophyll molecules in the plastids. 2. Since the carotenoids do not screen and light is necessary for chlorophyll formation, an effectiveness spectrum of protochlorophyll can be obtained which is the reciprocal of the light energy necessary to produce a constant amount of chlorophyll with different wavelengths. The relative effectiveness of sixteen spectral regions in forming chlorophyll was determined. 3. From the effectiveness spectrum, one can conclude that protochlorophyll is a blue-green pigment with major peaks of absorption at 445 mµ, and 645 mµ, and with smaller peaks at 575 and 545 mµ. The blue peak is sharp, narrow, and high, the red peak being broader and shorter. This differs from previous findings where the use of rougher methods indicated that red light was more effective than blue and did not give the position of the peaks of absorption or their relative heights. 4. The protochlorophyll curve is similar to but not identical with chlorophyll. The ratio of the peaks of absorption in the blue as compared to the red is very similar to chlorophyll a, but the position of the peaks resembles chlorophyll b. 5. There is an excellent correspondence between the absorption properties of this "active" protochlorophyll and what is known of the absorption of a chemically known pigment studied in impure extracts of seed coats of the Cucurbitaceae. Conclusive proof of the identity of the two substances awaits chemical purification, but the evidence here favors the view that the pumpkin seed substance, which is chemically chlorophyll a minus two hydrogens, is identical with the precursor of chlorophyll formation found in etiolated plants.     

Extrusion of a Protein-Pigment Complex from Blepharisma undulans

SYNOPSIS. Blepharisma undulans , a purple-coloured ciliate protozoan, contains pigment granules in the ectoplasm. Treating the cells with 0.9% NaCl extruded the pigment into the medium without injury to the cells. The extrusion was instantaneous and with a single treatment about 35% of the total pigment oozed out. The pigment in the cells as well as in the saline extract was found to be associated with proteins and particulate fractions. The protozoa regenerated the pigment when transferred to culture fluid but not in dilute solution of saline, where they continued to live without regenerating the pigment. In addition to NaCl, sulphates of monovalent and divalent cations and calcium-binding agents induced the extrusion, which, however, was prevented by calcium in all cases. It is proposed that the removal of calcium from the outer membrane by chemicals disintegrates the pellicle and produces the extrusion, since a majority of the pigment granules are in the subpellicular layers of the cell.     

Is Blepharisma hyalinum Truly Unpigmented?1

ABSTRACT. To answer whether Blepharisma hyalinum is truly unpigmented, the organism must be established in culture as pointed out by Giese in 1973. Accordingly, the present study deals with B. hyalinum kept in culture since its isolation in 1975. The organism still remains colorless after growth in the dark; however, it contains cortical granules resembling pigment granules in colored species. A comparative study was therefore undertaken of B. hyalinum and B. steini; both species have a compact macronucleus, though of different shape. Crude pigment was extracted with acetone from organisms grown in the dark for three weeks and the maxima were measured by absorption. Purified pigment was obtained from TLC-plate preparations and the absorption maxima were measured after removal of lipids with chloroform. No maxima characteristic of blepharismin were found in extracts of B. hyalinum, but these were present in extracts of B. steini. Electron microscopy of the cortical region revealed membrane-bound granules in both species; these granules differed in content but not in their capacity to extrude. In B. hyalinum all granules had a homogenous electron-dense substructure; in B. steini the granules had a net-like granulated substructure of varying electron density. This difference corresponds to that published on “pigment” granules in albino and pigmented strains of B. undulans. Our conclusions are that B. hyalinum is unpigmented (and a valid separate species) and that the cortical granules may serve other functions than that of storing blepharismin.     

Light maintains High Levels of Phytochrome Intermediates

THE plant photomorphogenetic pigment phytochrome exists in two forms, Pr and Pfr, interconvertible by light, which have peaks of absorption in the red and far-red regions of the spectrum respectively¹. Intermediates between Pr and Pfr have been demonstrated during photoconversion by Linschitz and his coworkers^2,3 using flash photolysis techniques. Low temperatures studies have also proved useful in identifying intermediates^4–8. Briggs and Fork^9,10 detected intermediates in vitro and in vivo in conditions of pigment cycling by mixed red and far-red light, but were restricted to studying the minor peaks of phytochrome absorption in the blue region of the spectrum because of the available instrumentation. In this type of measurement the problem is that actinic light has to be prevented from falling onto the photomultiplier. Briggs and Fork inserted a red cutoff filter, but this precluded measurement at the peaks of absorption of Pr and Pfr in the red and far-red regions of the spectrum. The design and construction of a sensitive quasi-continuous measuring spectrophotometer have now overcome this problem and made possible an investigation in vivo of phytochrome intermediates at any wavelength under conditions of pigment cycling, for example, in high intensity white incandescent light. The instrument can detect intermediates with a half life in excess of 0.2 ms. The longer lived intermediates between Pr and Pfr observed in the in vitro flash studies^2,3 should be readily detectable if they accumulate in conditions of cycling.     

Retardation of Division of Three Ciliates by Intermittent and Continuous Ultraviolet Radiations at Different Temperatures

The same dosage of ultraviolet (UV) radiation retards division of several protozoans more effectively when the light is intermittent than when it is continuous, and especially at temperatures of 25–35°C. At lower temperatures the difference between the effects of intermittent and continuous radiations is less marked. Somewhat similar results were obtained with the ciliates Paramecium caudatum, Blepharisma japonicum, and Colpidium colpoda, the disparity between intermittent and continuous light decreasing in the order given. The data are taken to indicate that thermochemical dark reactions succeed the absorption of UV radiations by the cells. In Blepharisma, besides initial delay in division, the cells stop dividing after one or two divisions, a "stasis" ensuing. Stasis is marked when the cells are irradiated at higher temperatures but is slight when they are irradiated at low temperatures, as if the temperature-sensitive reaction involved stasis (in all cases cultures are grown at 25°C). The data are related to findings in the literature.     

小蜡果皮水溶性色素的提取及光敏感性

以去离子水和不同体积分数乙醇从小蜡果皮中浸提红色素,用光谱扫描法检测该红色素的光吸收特性,比较不同浸提时间及不同体积分数乙醇对浸提效果的影响,并用暴露方式进行光敏感性测定。结果显示,小蜡果皮色素在纯水中的溶解性最好,属水溶性色素,延长浸提时间可提高色素的浸出率,但杂质质量分数也随之提高,紫外线对色素的色度影响最大,直接照射可使色素的色度大幅度下降。     

Pigment production from tryptophan by an Achromobacter species

Duerre, John A. (University of North Dakota, Grand Forks), and Patrick J. Buckley. Pigment production from tryptophan by an Achromobacter species. J. Bacteriol. 90:1686-1691. 1965.-A microorganism was isolated from the soil near the University of North Dakota. Biochemical and morphological characteristics indicated that this organism would best be classified as a member of the family Achromobacteraceae, genus Achromobacter, species unknown. The organism produced a red pigment when grown in a medium containing yeast extract and tryptophan. The pH optimum for pigment production was about 8.0 and the optimal temperature was 25 C. During a study of the nutritional requirements for growth and pigment production, it was found that the organism would grow and produce pigment in a medium containing tryptophan and nucleosides, but the rate of both growth and pigment formation in this medium was slower than that observed with tryptophan and yeast extract. The organism grew well in the presence of acid-hydrolyzed casein and nucleosides without producing pigment, indicating that the pigment is not necessary for growth. Resting-cell experiments definitely established tryptophan as the sole exogenous requirement for pigment production. The pigment was extracted from yeast extract-tryptophan medium with chloroform. Thin layer chromatographic analysis of the crude pigment extracted from this medium revealed the presence of two other pigments in addition to the major red pigment. One of these was a highly fluorescent orange pigment and the other a pink pigment. Only the red pigment was produced by resting cells in the presence of tryptophan alone. This pigment served as an electron acceptor when coupled with formic dehydrogenase, indicating its possible function as an oxidation-reduction pigment. The oxidized pigment had absorption peaks at 506 and 304 mmu. The peak at 506 mmu disappeared upon reduction with sodium sulfite. Shaking the reduced pigment in air proved to be an unsatisfactory method for returning the reduced pigment to the oxidized, colored state.     

地钱、拳卷地钱和粗裂地钱的紫外谱线组法鉴别

本文建立了紫外谱线组法鉴别地钱、拳卷地钱和粗裂地钱的方法。通过这三种药材的石油醚、氯仿、乙醇和水提取液的紫外谱线比较,发现地钱、拳卷地钱和粗裂地钱的紫外谱线图、最大吸收峰数目及峰位值具有明显差异。该法简单、准确,可用来鉴别地钱、拳卷地钱和粗裂地钱的原药材。     

Spectroscopic study of the chromophore--protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum.

Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores (blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicum's light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell (oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment-protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, free oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640--750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein acceptor residue as it had been suggested in previous studies.     

Effect of organic solvents on stability and activity of two related alcohol dehydrogenases: a comparative study.

A comparative study was performed regarding the catalytic activity and stability of two related enzymes (thermophilic alcohol dehydrogenase from Thermoanaerobacter brockii and its mesophilic counterpart from yeast) in the presence of a number of miscible and immiscible organic solvents. The study was performed in view of the practical usefulness of organic solvents for alcohol dehydrogenases which have been shown to catalyse a variety of industrially-important dehydrogenation reactions. A number of organic solvents of different physicochemical characteristics were used and substantial stabilization was achieved. The non-polar solvents utilized showed the ability to enhance thermal stability of both proteins. Protection against thermal denaturation was especially pronounced by n-dodecane, the solvent with the highest logP used in the present study. Dimethylformamide and dioxane, employed as two miscible organic solvents, showed the ability to cause substrate inhibition and changes in protein conformation as indicated by kinetic and fluorescence studies. A higher resistance of the thermophilic protein to the deleterious effect of pyridine and thermostabilization of the mesophilic enzyme by non-polar solvents are especially emphasized. Combined differences in protein structure and nature of organic solvents are suggested to explain the differences in stability and catalytic activity observed in the present investigation.