Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Sequential control of phytochrome-mediated synthesis de novo of β-amylase in the cotyledons of mustard (Sinapis alba L.) seedlings

In the cotyledons of mustard (Sinapis alba L.) seedlings irradiated from the time of sowing with continuous red light, the photoreversibility of the phytochrome-mediated increase in -amylase activity (EC 3.2.1.2) is lost 36 h after sowing (coupling point). However, the induced increase of -amylase activity cannot be detected before 46 h after sowing (starting point). Density labeling with deuterium oxide shows that the increase of enzyme activity in light and darkness coincides precisely with the synthesis of -amylase protein. Thus, phytochrome mediates an increase of -amylase synthesis de novo. Since there is no turnover detectable by density labeling, it is concluded that -amylase of mustard cotyledons is a physiologically stable enzyme (half-life >5 d). The 10-h time gap between loss of photoreversibility and onset of light-induced -amylase synthesis points to a relatively stable regulatory element within the signal chain (transmitter) which links -amylase synthesis to the primary action of phytochrome. A 12-h lag between the cessation of phytochrome action and the cessation of induced -amylase synthesis indicates a limited lifetime of the transmitter (about 12 h). The effect of this result on the interpretation of the coupling point is discussed.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome     

Production of deuterated β-carotene by metabolic labelling of Spirulina platensis

Method for production of deuterated -carotene for the bioavailability studies of vitamin A has been developed using Spirulina platensis in culture. Suspension cultures of Spirulina in heavy water (99.4% D₂O) medium produced maximum biomass and -carotene in 28 to 30 days. Of the total carotenoids, lutein constituted 30 to 35% while -carotene was about 24%. MS showed that 60 to 65% H atoms in -carotene were deuterated. 100% replacement of H atom with deuterium was achieved by preventing exchange with atmospheric moisture. The medium could be used in several cycles for metabolic labelling of carotenoids whereby the cost of production is reduced.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Unusual antiproliferative effects of transforming growth factors-β 1 andβ 2 against primary cells from human tumors

Transforming growth factors 1 and2 (TGF-1 and2), tested in a clonogenic assay against primary cells from human tumors, suppress proliferation to different extents. In nineteen of twenty-six cell cultures, proliferation was < 50% of control with factor at 0.04 or 0.4 nM. Of these, TGF- 2 was more active than TGF-1 in fourteen; and TGF-1 was more active than TGF-2 in five. In seven of the nineteen, proliferation was 0% with one or the other factor. In contrast, cisplatin was much less effective in inhibiting proliferation of some of the same cells even at 1,000 or more times the molar concentration of the factors. Surprisingly, when TGF- 1 and TGF-2 were combined at equal concentrations, the antiproliferative effect of one was cancelled or markedly inhibited by the other.     

Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Summary Plasmid-coded -glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The -glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the -glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of -glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK _m values for cellobiose and PNPG indicated that the -glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the -glucosidase fromE. coli pJS3 showed higher affinity for PNPG.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Water-soluble proteins of mature barley endosperm: genetic control,polymorphism, and linkage with β-amylase and spring/winter habit

Summary Water-soluble proteins (WSP-2 and WSP-3) and -amylase (-AMY-1) were extracted from mature endosperms of 44 spring and 39 winter barley genotypes. The protein and enzyme isoforms were separated in isoelectric focusing gels with a pH gradient of 4–6.5. The Wsp-3 and -Amy-1 loci were located to chromosomes 4H using the wheat/barley chromosome addition lines. Segregation analysis of F₂ and doubled haploid populations showed Wsp-2 and -Amy-1 to be tightly linked, with a map distance of 11 cMorgans. Isoforms of WSP-2 possessed similar pIs to that of WSP-3 and overlapping bands were observed in the gels. These bands segregated independently in F₂ and doubled haploid populations, implying two unlinked genes. All three loci were found to be polymorphic: two alleles were detected at the Wsp-2 locus, three at Wsp-3 and two at -Amy-1. The frequency of alleles at all three loci was found to be different in winter and spring genotypes. Spring genotypes possessed a wider range of phenotypes than winter genotypes. Spring and winter genotypes could be distinguished on the basis of WSP-3 and - AMY-1 phenotypes. The linkage between Wsp-3 and -Amy-1 loci and genes controlling spring/winter habit on chromosome 4H is discussed. It is concluded that Wsp-3 and -Amy-1 can be used as genetic markers for spring/winter habit in barley genetic research and breeding.     

Isozyme polymorphism ofβ-glucosidase inAspergillus nidulans

Electrophoretic analysis of the distribution of various electromorphs at different -glucosidase zones was carried out in natural populations ofA. nidulans, theA. nidulans group, and various species belonging to the genusAspergillus from diverse geographical areas of India. The data show the existence of three segregating zones for -glucosidase, designated -GluI, -GluII and -GluIII. All three zones are present in wild isolates ofA. nidulans, and only two, i.e., -GluI and -GluIII, in theA. nidulans group and -GluII and -GluIII in different species ofAspergillus exceptA. terreus, A. flavus, andA. brevipes, where only -GluIII is present. Overall nine electromorphs are observed at -GluI, three at -GluII, and six at -GluIII zones, respectively, It can be concluded that there may be three structural genes for -glucosidase coding the three polymorphic zones inA. nidulans.This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi.     

Antisense inhibition of the beta-carotene hydroxylase enzyme in Arabidopsis and the implications for carotenoid accumulation,photoprotection and antenna assembly

The xanthophylls are oxygenated carotenoids and are important structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex apoproteins (LHC) and contribute to photoprotection via non-photochemical quenching of chlorophyll fluorescence (NPQ) in oxygenic photosynthetic organisms. Previously, mutations have been described that disrupt many steps in the xanthophyll biosynthetic pathway. However, there are no definitive reports of a lesion that effects the -hydroxylase enzyme, which catalyzes hydroxylation of the -rings of -carotene and -carotene, and is thus necessary for synthesis of essentially all xanthophylls of higher plant chloroplasts. We have utilized an antisense approach to effectively reduce levels of -hydroxylase in Arabidopsis thaliana in order to examine how a reduction in this enzyme impacts carotenoid biosynthesis and plant viability. Expression of the antisense -hydroxylase transgene resulted in a maximal reduction in violaxanthin of 64% and a maximal reduction in neoxanthin of 41%. This reduction was reflected in a 22% increase in -carotene and a reduction in the total carotenoid pool, whereas lutein levels were relatively unaltered. Despite the reduction in violaxanthin and neoxanthin, the antisense -hydroxylase plants had a wild-type complement of chlorophylls and LHCs on a fresh weight basis. Under high light stress, the unconverted pool of violaxanthin was the same size as in wild type and thus there was an even greater proportional reduction in zeaxanthin of 75%. Despite this marked decrease in zeaxanthin, NPQ only declined by 16%.This revised version was published online in October 2005 with corrections to the Cover Date.     

Primary structures of alpha- and beta-subunits of alpha-amylase inhibitors from seeds of three cultivars of Phaseolus beans

The primary structures of three -amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits and from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, and , were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording -amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each -subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each -subunit of TAI and MAI-2 had two N-glycosylation sites, while the -subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each -subunit and M3FX at Asn(83) in -subunits of TAI and MAI-2.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

Characterization of Baboon (Papio hamadryas) Milk Proteins

The major proteins of baboon milk were identified as -lactoglobulin (LG), -lactalbumin (LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human LA, lysozyme, and albumin and bovine LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon LG are identical to those of macaque (Macaca fasicularis) LG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of LG were elucidated using RT-PCR amplification of poly(A)⁺ mRNA purified from lactating mammary gland. Baboon LG consists of 168 amino acid residues (M_r 20,750) and is the longest LG identified to date. LG and LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4–6, of individual baboon milk samples at varying stages of lactation.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

ζ-Carotene<Emphasis Type="Italic"> cis</Emphasis> isomers as products and substrates in the plant poly-<Emphasis Type="Italic">cis</Emphasis> carotenoid biosynthetic pathway to lycopene

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and -carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9-tricis--carotene with 15,9-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by -carotene desaturase, the 15-cis double bond of 9,15,9-tricis--carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9-dicis--carotene is utilized by -carotene desaturase via 7,9,9-tricis-neurosporene to 7,9,7,9-tetracis-lycopene. Other -carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis--carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to -carotene. The 5-cis-lycopene and the 9-cis-and 13-cis--carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.     

β-Xylanase production by Aureobasidium pullulans grown on sugars agricultural residues

Aureobasidium pullulans grew well in media containing glucose, fructose, xylan or xylose but -xylanase was only produced with xylan or xylose. Lactose and maltose were poor substrates for growth. -Xylanase production was repressed in media containing glucose or fructose along with xylose. Agricultural residues, such as wheat bran, paddy husk and rice straw, could be used as carbon sources for growth and -xylanase production of Aureobasidium pullulans. Tween 80 at 0.5% (v/v) increased the yield of -xylanase by up to 20%.     

A gene encoding Achlya bisexualis β-amylase and its expression in Saccharomyces cerevisiae

Part of a -amylase genomic DNA sequence from the oomycete, Achlya bisexualis was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotide primers derived from the conserved regions of other known -amylase sequences. The 5- and 3-regions of the -amylase gene were amplified by genome walking method. The Ach. bisexualis -amylase gene consisted of a 1338bp open reading frame, encoding a protein of 446 amino acids with a molecular weight of 49 381Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 67% similarity to the -amylase of Saprolegnia ferax, followed by 40% similarity to that ofArabidopsis thaliana. The -amylase gene was expressed in Saccharomyces cerevisiae placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.