Chlorophylle und Carotinoide der Chaetophorineae (Chlorophyceae; Ulotrichales)

In air-supplied inorganic liquid cultures, the highly differentiated green alga Fritschiella tuberosa forms only branched filaments with long slender cells. In nitrogen-deficient medium and with ageing of the cultures these cells become much shorter in length by subsequent formation of transverse walls. The chloroplasts of the slender cells contain the typical pigments of green algae. Together with the morphogenetic change to “short cells” secondary carotenoids are synthesized and stored in lipid droplets. Besides traces of lutein, violaxanthin and neoxanthin and reduced amounts of β-carotene, the following pigments have been demonstrated: Esters of astaxanthin (main pigment) and adonixanthin, canthaxanthin, echinenone and a Keto-α-carotinoid, which presumably is identical with α-doradexanthin, (3,3′-Dihydroxy-4-keto-α-Carotene) a pigment not known in plants until now. In nitrogen deficient cultures the chlorophylls are totally decomposed, the total-lipids increase by about 500%. By supplying nitrogen-deficient cultures with nitrogen and subsequent illumination regreening of the cells starts already 16 hrs later.     

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.     

Formation of stereoisomeric mixtures of naphthoquinone derivatives in Echium lycopsis callus cultures

Callus cultures of Echium lycopsis were shown to produce a large amount of a mixture of red pigments consisting of five esterified derivatives of 5,8-dihydroxy-2-(1-hydroxy-4-methyl-3-pentenyl)-1,4-naphthoquinone. Examination of the absolute configuration of these compounds revealed that the cultures produced both the R-form (shikonin) and the S-form (alkannin) in various ratios depending upon the esterified derivative, although the overall ratio for the total derivatives was ca 1:1. On the other hand, all the corresponding derivatives produced by Lithospermum cultures were primarily of the R-form. It was also demonstrated that pigment formation in Echium cultures was inhibited by either white or blue light as well as by the synthetic auxin 2,4-D as in the case of Lithospermum cultures.     

Synthesis of photopigments and electron transport components in synchronous phototrophic cultures of Rhodopseudomonas sphaeroides.

The kinetics of synthesis and incorporation of the photosynthetic pigments and several of the major oxidative and photosynthetic electron transport components of Rhodopseudomonas sphaeroides have been studied during synchronous and asynchronous phototrophic growth. The photosynthetic pigments and cytochromes c and b, measured spectroscopically, exhibited continuous patterns of synthesis and incorporation into the membrane particulate fraction in both synchronous and asynchronous cultures. Succinic dehydrogenase and NADH-oxidase activities, present at low levelnous growth. In a previous paper, Leuking, D.R., Fraley, R.T., and Kaplan, S. ((1978) J. Biol. Chem. 253, 451-457) have shown that total cellular phospholipid is also accumulated discontinuously during synchronous growth. A continuously incorporated membrane component is thus subject to a wide variation in the membrane protein/lipid ratio. The significance of this ratio in regulating the activity of membrane proteins is discussed and the distinction between protein incorporation and function is drawn with particular reference to the photosynthetic pigments and cytochrome components and the oxidative activities measured. It is suggested that a dependence of membrane protein activity on the membrane protein to lipid ratio in vivo is of possible significance in the control of membrane synthesis and cell division.     

Senescence-Related Changes in the Lipid Transition Temperature of Microsomal Membranes from Algae

The phase behaviour of smooth microsomal membranes from senescing cultures of Scenedesmus quadricauda has been examined by wide-angle x-ray diffraction. The algae were grown in Bristol's medium at 22°C under continuous illumination. The transition temperature, taken to be the highest temperature at which crystalline (gel) phase lipid can be detected, increased with culture age from a low of 0°C for young cultures to a high of about 70°C for 140-day-old cultures. This indicates that for young cultures the membrane lipid is entirely liquid-crystalline (fluid) at physiological temperatures, but as the cultures age portions of the lipid become crystalline. The increase in transition temperature showed a close temporal correlation with loss of chlorophyll and loss of protein per g dry weight, and can thus be construed as an index of senescence. The unsaturated to saturated fatty acid ratio of the membrane lipid, while fluctuating with culture age, did not show any consistent trend that could be related to the change in transition temperature. Thus the formation of gel phase lipid does not appear to be due to a change in fatty acid saturation.     

Characterization of an hyperpigmenting mutant of Monascus purpureus IB1: identification of two novel pigment chemical structures

Monascus purpureus IB1 produces about 50-fold higher levels of azaphilone pigments than M. purpureus NRRL1596. Differently pigmented mutants were obtained from M. purpureus IB1 by nitrosoguanidine treatment. A highly pigmented strain, M. purpureus HP14, was found to lack the formation of the classical yellow and orange azaphilones and was found to produce only about 10% of the red azaphilone pigments. The intense color was associated with novel pigments as shown by high-performance liquid chromatography (HPLC). The addition of hexanoic acid to M. purpureus IB1 resulted in higher volumetric and specific red pigment productivity, but in a complete absence of the classical orange azaphilones, while the classical yellow and red azaphilone pigments were severely reduced; new peaks corresponding to less hydrophobic pigments were found in hexanoic-supplemented cultures by HPLC. Purification of pigments from hexanoic-supplemented cultures showed the presence of five new pigments as indicated by the absorption spectra and HPLC analysis. Two of them, R3 and Y3, were characterized by nuclear magnetic resonance as 9-hexanoyl-3-(2-hydroxypropyl)-6a-methyl-9,9a-dihydro-6H-furo[2,3-h]isochromene-6,8(6aH)-dione and 4-[2,4-dihydroxy-6-(3-hydroxybutanethioyloxy)-3-methylphenyl]-3,4-dihydroxy-3,6-dimethylheptanoic acid. These pigments were also found to be present in cultures of the high-producing mutant M. purpureus HP14. These new pigments are less hydrophobic than the classical azaphilones and may have better properties as natural colorants in the food industry.     

Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.     

Organisation of xanthophyll pigments lutein and zeaxanthin in lipid membranes formed with dipalmitoylphosphatidylcholine

Carotenoid pigments and in particular xanthophylls play several physiological functions in plant and animal membranes. Xanthophylls are present in biological membranes in the form of pigment-protein complexes but also as direct components of lipid phase. The biological activity of carotenoids in membranes depends on a molecular organisation of pigments in lipid bilayers, in particular the localisation, orientation and aggregational state. In the present work the organisation of lutein- and zeaxanthin-containing lipid membranes was analysed with the application of electronic absorption spectroscopy. Both xanthophyll pigments incorporated to the dipalmitoylphosphatidylcholine (DPPC) unilamellar liposomes form H-type molecular aggregates, manifested by the hypsochromic shift of the main absorption band of carotenoids. The aggregation of lutein and zeaxanthin in DPPC membranes was observed even at relatively low concentrations of a pigment in the lipid phase (1-5 mol%). Gaussian analysis of the absorption spectra of lutein and zeaxanthin in DPPC membranes in terms of the exciton splitting theory revealed the formation of different molecular structures of pigments interpreted as dimers, trimers, tetramers and large aggregates. The fraction of lutein and zeaxanthin in the monomeric form was found to depend on the physical state of the lipid phase. Pronounced monomerisation of lutein and zeaxanthin was observed as accompanying the transition from the P(beta)' phase to the L(alpha) phase of DPPC, mostly at the expense of the trimeric and tetrameric forms. The fraction of monomers of lutein is always lower by 10-30% than that of zeaxanthin under the same experimental conditions. Different organisational forms of lutein and zeaxanthin in the model system studied are discussed in terms of possible physiological functions of these pigments in the membranes of the retina: zeaxanthin in the protection of the lipid phase against oxidative damage and lutein in absorbing short wavelength radiation penetrating retina membranes.     

Microsomal lipid peroxidation: mechanisms of initiation. The role of iron and iron chelators

The role of iron and iron chelators in the initiation of microsomal lipid peroxidation has been investigated. It is shown that an Fe3+ chelate in order to be able to initiate enzymically induced lipid peroxidation in rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with rat liver microsomes has to fulfill three criteria: (a) reducibility by NADPH; (b) reactivity of the Fe2+ chelate with O2; and (c) formation of a relatively stable perferryl radical. NADH can support lipid peroxidation in the presence of ADP-Fe3+ or oxalate-Fe3+ at rates comparable to those obtained with NADPH but requires 10 to 15 times higher concentrations of the Fe3+ chelates for maximal activity. The results are discussed in relation to earlier proposed mechanisms of microsomal lipid peroxidation.     

The Effect of Antihistamines on the Pigments of Green Protista.

SUMMARY. A preliminary survey of the influence of a number of antihistamines on growth of several protozoa indicated that not only growth but also pigment formation in green forms was inhibited. Subsequently, it was demonstrated that molar concentrations of the drugs of less than 1 times 10⁻³ caused chlorosis of cultures of Euglena gracilis var. bacillaris, Chlamydomonas pseudococcum , and Chlorella vulgaris.
With the aid of one of the antihistamines, Pyribenzamine (CIBA), a permanently colorless culture of Euglena was obtained. This culture has been maintained in the chlorotic state in antihistamine-free medium through eighteen serial transfers in constant light. Comparative spectro-photometric studies were made of the pigments extracted from this new euglenid strain with those from normal green, dark-grown and streptomycin-bleached euglenas. Although the dark-etiolated forms retained small amounts of chlorophylls, neither of the drug-bleached cultures showed evidence of these pigments. Quantitatively, the carotenoid picture in dark-bleached and streptomycin-grown organisms was similar, but these pigments were greatly reduced in the Pyribenzamine-treated strain. Further studies on the identity of the carotenoids in the new euglenid are in progress.     

Long-term culture of human bone marrow macrophages: macrophage development is associated with the production of granulomonopoietic enhancing activity (GM-EA)

We describe a liquid culture system allowing the long-term maintenance and differentiation of human marrow monocytes into well-developed, lipid-containing macrophages, and we show that these cells produce a regulating activity that enhances the colony formation induced by granulocyte-macrophage colony-stimulating factors (GM-CSF). Adherent, low density (less than 1.074 g/cm3) human marrow cells were used as a source of monocytic cells. Of 12 different culture conditions tested, alpha-medium supplemented with 20% horse serum and incubation at 37 degrees C provided the best conditions for macrophage development, adipogenesis, and long-term culture. Neither insulin (1 to 10 micrograms/ml) nor hydrocortisone (10(-6) M) improved the lipid accumulation in cultures containing horse serum. Trypsin was employed to remove fibroblasts without detaching monocytic cells from the marrow-derived adherent cell layers. Marked structural and functional changes characterized the transformation of monocytes into lipid-containing macrophages. Cell enlargement up to seven or eight times by 21 to 28 days of culture was associated with an increase in small and medium-sized lipid granules, as well as in acid phosphatase and nonspecific esterase activities. Ultrastructurally, there was an increase in the number of mitochondria, Golgi apparatus, lysosomes, rough endoplasmic reticulum, and lipid inclusions, which remained of small size and did not coalesce to form larger inclusions. The absolute quantity of Fc receptors, Ia antigens, and antigens recognized by monoclonal antibodies 61D3 and 63D3 also increased as a function of cell size. As marrow monocyte-macrophage differentiation proceeded, a rapid decline in GM-CSF production was accompanied by an increase in an activity that by itself had no capacity to stimulate colony formation in CFU-GM cultures devoid of GM-CSF, but did enhance the colony formation induced by optimal concentrations of GM-CSF. Neither the enhancing activity nor its production was related to the horse serum present in the culture supernatants. The morphology of colonies enhanced by this activity was different from the morphologic spectrum in non-enhanced cultures. This granulomonopoietic enhancing activity (GM-EA) represents another positive feedback regulator of hematopoiesis derived from cells of the monocyte-macrophage system.     

Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation

The green microalga Neochloris oleoabundans is able to grow in both low and high salinity media and is largely studied for its capability to accumulate lipids under starvation. Moreover, N. oleoabundans is a mixotrophic alga, and then organic carbon addition can promote its growth. This research aims to study the morpho-physiological aspects, with a particular attention on the photosynthetic response, both during mixotrophic growth and starvation in brackish media, more sustainable than freshwater cultivation. In the first step, the alga was cultivated mixotrophically in a brackish medium added with an apple waste product; in the second one, cells were starved also to verify lipid induction. Results indicate that growth is highly promoted during the first week of mixotrophic cultivation, while photosynthetic pigments and lipids are over-produced during the following three weeks of cultivation. In parallel, in mixotrophic cultures the maximum PSII quantum yield was enhanced during the exponential phase of growth. Interesting changes affected the mixotrophic cultures with respect to the partitioning of absorbed light energy. Starvation of both 7-day-grown mixotrophic and autotrophic cultures caused growth inhibition, pigments and photosynthesis downshifting, and concomitantly promoted evident lipid synthesis.     

Relationship Between Age of Culture and Occurrence of the Pigments of Photosystem II of Photosynthesis in Heterocysts of a Blue-Green Alga

Microspectrophotometric examination of the pigments in vivo of heterocysts of Anabaena sp. L-31 has shown that most heterocysts of 2-day-old cultures possess only very small amounts, if any, of c-phycocyanin, allo-phycocyanin, and c-phycoerythrin, the main pigments comprising photosystem II of photosynthesis. The quantities of these pigments, however, increase with age of cultures, and by the end of 5 days the majority of heterocysts contain comparatively large amounts. The culmination of this sequential development is observed in most heterocysts of 7- to 15-day-old cultures when the full complement of photosystem II pigments is present. The spectral characteristics at this stage are similar to those of vegetative cells and suggest a dedifferentiation of heterocysts.     

An improved method for the rapid and easy separation of leaf pigments and their derivatives by thin-layer chromatography

A thin-layer chromatographic method for the separation and identification of leaf pigments and their degradation products on commercial silica gel layer has been developed to give a tool to examine the purity of chlorophyll preparations and the chemical stability of chlorophyll molecules during the course of the chlorophyll preparation. It has been confirmed that the developing solvent system (isopentane:tert-butyl alcohol:acetone = 90:5:5, v/v/v) is quite useful to separate the photosynthetic pigments and their degradation products which were commonly found during the course of in vitro chlorophyll studies.     

Monitoring growth and lipid production of new isolated oleaginous yeast Cryptococcus aerius UIMC65 on glucose and xylose cultures

Process monitoring is one of the most important factors affecting production efficiency at industrial scale bioprocesses. In the present work, Flow-cytometric analysis has been employed to monitor and determine neutral lipid cell droplets, granularity and size of the cells of the new oleaginous yeast, Cryptococcus aerius UIMC65. It has been shown that, differences of fluorescent intensity as well as side and forward scatter light properties have close correlations with the differences in lipid production by these yeast cells. The lipid content-related fluorescent intensity versus forward scatter parameter has been used to monitor and compare different subpopulations during growth phases on both glucose and xylose in batch cultures. Flow cytometric results have revealed that the observed differences in the proportion of each subpopulation were related to the specific growth phase and lipid content of the cells. The highest lipid content and lipid productivity were attained at 82.62%, 4.47 g/L (at 72 h) and 78.41%, 6.21 g/L (at 60 h) on glucose and xylose growth cultures, respectively. The highest biomass, lipid yield and biomass yield were found to be 7.92 g/L (on glucose culture, at 60 h), 20.92% (on glucose culture, at 48 h) and 50.71% (on glucose culture, at 24 h), respectively.     

Characterization of akinete differentiation in the cyanobacterium Anabaena azollae

Differentiation of akinetes was investigated in the filamentous cyanobacterium Anabaena azollae Stras. In this organism all pre-existing vegetative cells are capable of developing into akinetes. Standard sporulation medium (SSM) was used to synchronously induce the formation of akinetes, while cultures in Allen and Arnon (AA/8) medium were used as controls.This paper describes the changes in photosynthetic pigments and total soluble proteins in these cultures over a 25-day period encompassing akinete differentiation. Heterocyst frequencies and nitrogenase activity were also monitored during the same period in both media. SDS-PAGE results indicated that specific proteins were synthesized in a manner correlated with akinete differentiation. The results demonstrate that in cultures undergoing akinete development, some of the photosynthetic pigments are maintained, nitrogen-fixation and heterocyst differentiation are suppressed, and the cells synthesize a variety of specific proteins.     

Einfluß von Eisen und Sauerstoff auf die Pigmentbildung bei verschiedenen Pseudomonas-Spezies

Zusammenfassung Die Bildung von Pigmenten mit Redoxfunktion in Pseudomonas-Kulturen ist vom Eisengehalt des Nährbodens abhängig. In belüfteten Kulturen war die Menge der abgelagerten Farbstoffe stark herabgesetzt. Die bisher untersuchten Pigmente waren: Phenazin--carbonsäure aus Pseudomonas aureofaciens, Pseudoiodinin aus Ps. fluorescens var. pseudoiodinum und Pyocyanin aus Ps. aeruginosa.

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Effect of iron and oxygen on the formation of pigments in some Pseudomonas spp.

Summary The formation of pigments having the function of redox catalysts in cultures of Pseudomonas spp. with gluconate as a sole carbon source was found to be related to the concentration of iron in the medium. In aerated cultures the accumulation of these pigments was extremely decreased.These pigments were: phenazine--carboxylic acid formed by Pseudomonas aureofaciens, pseudoiodinine formed by Ps. fluorescens var. pseudoiodinum, and pyocyanine formed by Ps. aeruginosa.

     

Outdoor H₂ production in a 50-L tubular photobioreactor by means of a sulfur-deprived culture of the microalga Chlamydomonas reinhardtii

In the past decade, H? production using the green microalga Chlamydomonas reinhardtii has been extensively studied under laboratory-scale photobioreactors, while information on outdoor cultures is still lacking. In this paper, the results of experiments conducted with sulfur-deprived cultures of C. reinhardtii carried out in a 50-L horizontal tubular photobioreactor are presented. Hydrogen production experiments were carried out under both artificial and direct solar light. In both cases, the H? output attained was 18-20% of what obtained in the laboratory. However, no significant changes in the H? production were observed when cells grown outdoors were tested under laboratory conditions. Chlorophyll fluorescence measurements showed that outdoor cultures were subjected to strong photo-inhibition, due to the combination of high solar light intensity and sulfur-deprivation. Indeed, H? production was only achieved outdoors when cultures were previously acclimated to sunlight, a condition that caused a number of physiological changes, namely: (i) a decrease in the chlorophyll content per unit of dry weight; (ii) an increase in the photosynthesis and respiration rates, and (iii) a higher induction of the xanthophyll cycle pigments as compared to non-acclimated cultures. It was concluded that the reduced H? output achieved in the 50-L photobioreactor was due to the different illumination pattern to which the cultures were exposed (one-sided vs. two-sided illumination provided in the laboratory), as well as to the great difference in the mixing times (60 min vs. 15.5s achieved in the lab-scale photobioreactor). To the very best of our knowledge this is the first time that H? production with green algae has been achieved by means of solar light.     

Interactions between canthaxanthin and lipid membranes — possible mechanisms of canthaxanthin toxicity

Canthaxanthin (β, β-carotene 4, 4′ dione) is used widely as a drug or as a food and cosmetic colorant, but it may have some undesirable effects on human health, mainly caused by the formation of crystals in the macula lutea membranes of the retina. This condition is called canthaxanthin retinopathy. It has been shown that this type of dysfunction of the eye is strongly connected with damage to the blood vessels around the place of crystal deposition. This paper is a review of the experimental data supporting the hypothesis that the interactions of canthaxanthin with the lipid membranes and the aggregation of this pigment may be the factors enhancing canthaxanthin toxicity towards the macula vascular system. All the results of the experiments that have been done on model systems such as monolayers of pure canthaxanthin and mixtures of canthaxanthin and lipids, oriented bilayers or liposomes indicate a very strong effect of canthaxanthin on the physical properties of lipid membranes, which may explain its toxic action, which leads to the further development of canthaxanthin retinopathy.