ACCUMULATION OF β-CAROTENE IN HALOTOLERANT ALGE: PURIFICATION AND CHARACTERIZATION OF β-CAROTENE-RICH GLOBULES FROM DUNALIELLA BARDAWIL (CHLOROPHYCEAE)1

Dunaliella bardawil Ben-Amotz & Avron, but not most other Dunaliella species, has a unique property of being able to accumulate, in addition to glycerol, large amounts of β-carotene when cultivated under appropriate conditions. These include high light intensity, a high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under conditions of maximal carotene accumulation D. bardawil contains at least 8% of its dry weight as β-carotene while D. salina grown under similar conditions contains only about 0.3%. Electron micrographs of D. bardawil grown under conditions of high β-carotene accumulation show many β-carotene containing globules located in the interthylakoid spaces of the chloroplast. The same algae grown under conditions where β-carotene does not accumulate, contain few to no β-carotene globules. The β-carotene-rich globules were released from the algae into an aqueous medium by a two-stage osmotic shock technique and further purified by centrifugal ion on 10% sucrose. The isolated purified globules were shown by electron microscopy to be free of significant contamination and composed of membrane-free osmiophilic droplets with an average diameter of 150 nm. Reversed phase high performance liquid chromatography of a total pigment extract of the cells revealed the presence of β-carotene as the major pigment, together with chlorophylls a and b, α-carotene and the xanthophylls lutein, neoxauthin and zeaxanthin. β-Carotene accounted for essentially all the pigment in the purified globules. Analysis of the algal and globule β-carotene fractions by HPLC showed that the β-carotene was composed of approximately equal amounts of all-trans β-carotene and of its 9-cis isomer. Intact D. bardawil cells contained on a dry weight basis about 30% glycerol, 30% protein, 18% lipid, 11% carbohydrate, 9%β-carotene and 1% chlorophyll. The β-carotene globules were composed of practically only neutral lipids, more than half of which was β-carotene. It is suggested that the β-carotene globules may serve to protect D. bardawil against injury by the high intensity irradiation to which this alga is usually exposed in nature.     

PRODUCTION AND SELECTION OF HIGH β-CAROTENE MUTANTS OF DUNALIELLA BARDAWIL (CHLOROPHYTA)1

We describe a procedure for the selection of β-carotenerich mutants of the halotolerant alga Dunaliella bardawil Ben-Amotz & Avron. Under normal growth conditions the isolated mutants had a several-fold higher content of β-carotene than the wild type. Under carotene-induction conditions, the mutants also possessed a higher β-carotene content than the wild type. Both the production rate of phytoene and the conversion rate of phytoene to lycopene and β-carotene were accelerated in the mutants. Cycloheximide, which (in the wild type) inhibits the inductive synthesis of the proteins required for β-carotene production, had a much smaller effect on β-carotene biosynthesis in the mutants. We suggest that the mutants are affected in the regulatory path, which controls the induction of high β-carotene production in Dunaliella.     

THE WAVELENGTH DEPENDENCE OF MASSIVE CAROTENE SYNTHESIS IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE)1

Dunaliella bardawil Ben-Amotz & Avron accumulates high concentrations of β-carotene when grown under high light intensity. The β-carotene is composed mainly of 9-cis and all-trans β-carotene. Accumulation of β-carotene and an increase in the ratio of the 9-cis to the all-trans isomer are strongly dependent on the light intensity under which the algae are cultivated but are independent of light quality within the photosynthetically active radiation range. Cells grown under continuous red (>645 nm) or white light of 500 W·m^?2 reach a value of about 32 pg β-carotene·cell^?1 and a ratio of 9-cis to all-trans β-carotene of around 2, whereas cells grown under low red or white light intensity of 25 W·m^?2 contain about 3 pg·cell^?1 and a ratio of isomers of around 0.3.     

LOW-TEMPERATURE-INDUCED SYNTHESIS OF α-CAROTENE IN THE MICROALGA DUNALIELLA SALINA (CHLOROPHYTA)

The microalga Dunaliella salina (Teo.) is well known as an accumulator of β-carotene (β,β-carotene) when subjected to growth-limiting conditions (e.g. exposure to high irradiances). In addition, the carotenoid α-carotene (β,ε-carotene) may also be synthesized and subsequently accumulated by this alga under specific growth conditions. The main factor in stimulating the synthesis of this carotene was determined to be exposure to lower than optimum temperatures for algal growth. A 7.5-fold increase in the levels of α-carotene was observed when the temperature was decreased from 34 to 17° C, whilst levels of β-carotene were unaltered. The accumulation of α-carotene was unaffected by irradiance, although its isomeric composition was greatly altered by light levels. The proportion of 9- cis α-carotene increased from 15% to 45% of total α-carotene when the irradiance was decreased from 260 to 50 μmol·m⁻²·s⁻¹. Exposure to higher irradiances had little influence on the isomeric composition of this carotenoid. A reduction in growth temperature did not influence the isomeric composition of α-carotene. Nutrient status (nitrogen and phosphate) had no effect on either the content or isomeric composition of α-carotene accumulated by D. salina.     

ANALYSIS OF THE EXPRESSION OF GENES FOR P-TYPE ATPASE IN THE HALOTOLERANT ALGA DUNALIELLA SALINA (CHLOROPHYCEAE)1

A partial complementary DNA (cDNA) (DSA8) for a P-type ATPase was obtained from the halotolerant alga Dunaliella salina (Dunal) Teod. (Chlorophyceae). The cDNA exhibited greater than 90% homology to the cDNA for a H⁺-ATPase in D. bioculata Butcher. The expression of the gene that corresponded to DSA8 was decreased strongly by increases in NaCl concentration. The expression of a gene that corresponded to another ATPase (DSA1; possibly for a Ca²⁺-ATPase) from D. salina did not show the same decrease as did the DSA8. However, increased osmotic pressure due to glycerol resulted in the same decrease in the DSA8 gene. Under salt or osmotic stress, the activity of a H⁺-ATPase from microsomes of this alga also decreased. We suggest that expression of the gene for the plasma membrane H⁺-ATPase of D. salina is regulated by osmotic pressure rather than by the concentration of NaCl.     

THE GLYCEROL PERMEABILITY OF THE PLASMALEMMA OF THE HALOTOLERANT GREEN ALGA DUNALIELLA PARVA (VOLVOCALES)1

The glycerol permeability of the plasmalemma of the green alga Dunaliella parva Lerche was investigated by efflux studies with labelled glycerol, by enzymatic determination of glycerol leakage, and the determination of the reflection coefficient from osmotically induced volume changes (zero flow method). All results indicate that the plasmalemma of D. parva does not exhibit a special low permeability towards glycerol as would be expected from a glycerol accumulating alga. Rather, significant amounts of glycerol diffuse continuously into the medium following the glycerol concentration gradient between the cells and the medium. Efflux rates vary between 0.1 and 2 μmoles glycerol·mg^?1 chlorophyll·h^?1 depending on the external NaCl concentration. After one day up to 25% of the total glycerol of the algal suspension was found in the medium. Within 10 days this value can increase to 60%, depending on the growth constant of the culture. The reflection coefficient σ was determined to be 0.87, the permeability coefficient 2800 × 10^?11 m·sec^?1. To maintain a proper endogenous glycerol level corresponding to the external osmotic pressure, glycerol efflux in D. parva has to be balanced by a continuous synthesis of glycerol. D. parva follows the strategy of “glycerol efflux tolerance” instead of “glycerol efflux avoidance”. The alga has to pay the energetic costs of this strategy of tolerance.     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phytoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low β-carotene and in the norflurazon-treated phytoene-rich cells. These observations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil protects the cells against injury by excessive irradiation.     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phqtoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low & carotene and in the nor-urazon-treated phytoene-rich cells. These obseruations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil portects the cells against injury by excessive irradiation.     

A SALT-INDUCED CHLOROPLAST PROTEIN IN DUNALIELLA SALINA (CHLOROPHYTA)1

The unicellular green alga Dunaliella salina Teod, is halophilic and wall-less. The cell acclimates to osmotic stresses by accumulation or degradation of glycerol. To investigate other mechanisms involved in its physiological recovery following hyperosmotic shocks, protein profiles from cells grown in various salinities were compared. A 13-kDa protein (P13) accumulated when cells were subjected to drastic hyperosmotic shock. Front our results with antibiotic-treated cells and purified chloroplasts, we believe that this component results from de novo translation in chloroplasts. The solubility of P13 was strongly promoted by Triton X-100. Its accumulation was correlated with the recovery of photosynthesis.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE,AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α‐glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane‐bound isoforms, being more abundant as a membrane‐bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion‐exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α‐amylases and α‐galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α‐amylases acting on maize seed starch granules.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.