Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of β-carotene when grown outdoors under defined conditions. The β-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans β-carotene and 40% 9-cis β-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of β-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of β-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.     

The Biosynthesis of {beta}-Pyrazol-l-ylalanine

ß-Pyrazol-I-ylalanine, an isomer of histidine, occursin large amounts in several cucurbitaceous species. Enzymicsynthesis of the new amino-acid is shown to occur by the condensationof pyrazole and serine in an analogous manner to that in whichtryptophan is synthesized from indole and serine. The propertiesand distribution of the new enzyme, called ß-pyrazol-I-ylalaninesynthetase, have been studied using crude extracts of cucumberseedlings. The enzyme has also been demonstrated in extractsof other cucurbit seedlings. A chemical synthesis of ß-pyrazol-I-ylalanine fromserine and pyrazole adapted from the enzymic pathway has beenused to demonstrate indirectly the presence of pyrazole in cucumberand melon seeds.     

The Biosynthesis of Abscisic Acid from all-trans-{beta}-Carotene in a Cell-Free System from Citrus sinensis Exocarp

A cell-free system prepared from exocarp of Citrus sinensisfruits converted [¹⁴C]-all-trans-ß-carotene into zeaxanthin,antheraxanthin, violaxanthin and abscisic acid (ABA). ABA wasunequivocally characterized by combined capillary GC-MS. (Received August 3, 1993; Accepted August 6, 1993)     

Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil

Dunaliella bardawil, a unicellular microalga, grows in relatively high concentrations of salt and has so far been refractory to Agrobacterium-mediated transformation. An inverse relationship between salt concentration and hygromycin resistance was observed. Co-cultivation at 0.2?M NaCl allowed growth of both D. bardawil and A. tumefaciens. Lowering salt concentrations also enabled the use of lower concentrations of hygromycin, the selection agent. Cells resistant to 100?mg?l^?1 hygromycin were selected and growth of Agrobacterium was completely eliminated in these cells using cefotaxime/potassium clavulanate. The concentration of sodium chloride was gradually increased to 1.0?M with simultaneous reduction of hygromycin concentration for better growth of D. bardawil. Agrobacterium was unable to survive in the growth medium used for Dunaliella. Expression of β-glucuronidase (uidA), green fluorescent protein (GFP) and hygromycin phosphotransferase (hpt) in the hygromycin-resistant culture was detected using X-gluc as substrate and Western blotting using GFP antibodies and RT-PCR respectively. Cells growing in 1.0?M NaCl (in the absence of hygromycin) retained their ability to grow in hygromycin even after 18 months of cultivation. These cells expressed GFP and PCR for hpt gene was positive. The stability of the integrated transgene and resistance to hygromycin in three different transformation events were ascertained periodically. Southern blotting of DNA extracted from hygromycin resistant cells (HRC) that were 15–18 months old established the presence of the integrated transgene in the DNA of D. bardawil. Results of the present study substantiate A. tumefaciens-mediated transformation of the unicellular marine alga D. bardawil. Agrobacterium tumefaciens-mediated transgene integration along with the massive outdoor cultivation methods used for D. bardawil may allow the commercial synthesis of secondary metabolites and heterologous proteins.     

{beta}-Naphthyl oligophosphates: Inhibitors of photophosphorylation and H+-ATPase of spinach chloroplasts

ß-Naphthyl di-, tri- or tetraphosphate inhibits photophosphorylationof spinach chloroplasts competitively with ADP, whereas ß-naphthylmonophosphate inhibits it competitively with Pi. The apparentKi of ß-naphthyl diphosphate for the ADP site was300 µM and that of ß-naphthyl monophosphatefor the Pi site was 1.45 mM. At 10 mM, both of these two organicphosphates inhibited photophosphorylation more than 90%. Noneof the above four ß-naphthyl phosphates were phosphorylatedby chloroplasts. ß-Naphthyl di-, tri- or tetraphosphateinhibits ATPase activity of isolated chloroplast coupling factor1 (CF₁) (EC 3.6.1.3 [EC] ) and light-triggered ATPase activity ofchloroplasts competitively with ATP, whereas ß-naphthylmonophosphate acts non-competitively. None of the four ß-naphthylphosphates were hydrolyzed by these two ATPase activities. Atconcentrations equal to ADP or ATP, ß-naphthyl di-,tri- or tetraphosphate inhibited these three reactions in theorder; ATPase of isolated CF₁> photophosphorylation>light-triggeredATPase of chloroplasts. The results suggest that the effect of the monophosphate isprincipally on the Pi site(s) and that of the di-, tri- or tetraphosphateis on the adenine nucleotide site(s) on the active center ofCF₁. ¹Part of this work was reported at the 1979 Annual Meeting ofthe Japanese Society of Plant Physiologists (Nagoya, April 7,1979) and the 52nd Annual Meeting of the Japanese BiochemicalSociety (Tokyo, October 7, 1979). This work was supported inpart by Grants-in-Aid for Scientific Research from the Ministryof Education, Science and Culture, Japan (311808 and 311909). (Received November 14, 1979; )     

A Note on the Presence of {beta}-Amylase in Moulds

Evidence is given for the presence, in the mould Mucor Rouxianusand in several Rhisopus species, of an amylolytic enzyme producingglucose directly from starch. This glucosidase probably possessesa wide specificity range and attacks maltose as well. It doesnot destroy the iodine reaction of starch, but as a rule itis accompanied by another amylase which does destroy thb reaction.Of the mould amylases investigated, Mucor-amylase containedthe highest proportion of the glucosidase.     

Sequence determinants of enhanced amyloidogenicity of Alzheimer A{beta}42 peptide relative to A{beta}40

Aggregation of proteins into insoluble deposits is associated with a variety of human diseases. In Alzheimer disease, the aggregation of amyloid beta (Abeta) peptides is believed to play a key role in pathogenesis. Although the 40-mer (Abeta40) is produced in vivo at higher levels than the 42-mer (Abeta42), senile plaque in diseased brains is composed primarily of Abeta42. Likewise, in vitro, Abeta42 forms fibrils more rapidly than Abeta40. The enhanced amyloidogenicity of Abeta42 could be due simply to its greater length. Alternatively, specific properties of residues Ile(41) and Ala(42) might favor aggregation. To distinguish between these two possibilities, we constructed a library of sequences in which residues 41 and 42 were randomized. The aggregation behavior of the resulting sequences was assessed using a high throughput screen, based on the finding that fusions of Abeta42 to green fluorescence protein (GFP) prevent the folding and fluorescence of GFP, whereas mutations in Abeta42 that disrupt aggregation produce green fluorescent fusions. Correlations between the sequences of Abeta42 mutants and the fluorescence of Abeta42-GFP fusions in vivo were confirmed in vitro through biophysical studies of synthetic 42-residue peptides. The data reveal a strong correlation between aggregation propensity and the hydrophobicity and beta-sheet propensities of residues at positions 41 and 42. Moreover, several mutants containing hydrophilic residues and/or beta-sheet breakers at positions 41 and/or 42 were less prone to aggregate than Abeta40 wherein these two residues are deleted entirely. Thus, properties of the side chains at positions 41 and 42, rather than length per se, cause Abeta42 to aggregate more readily than Abeta40.     

Shortened {beta}-cell lifespan leads to {beta}-cell deficit in a rodent model of type 2 diabetes

Since the fundamental defect in both type 1 and type 2 diabetes is β-cell failure, there is increasing interest in the capacity, if any, for β-cell regeneration. Insights into typical β-cell age and lifespan during normal development and how these are influenced in diabetes is desirable to realistically establish the prospects for β-cell regeneration as means to reverse the deficit in β-cell mass in diabetes. We assessed the mean β-cell age and lifespan by the classical McKendrick-von Foester equation that describes the age-based heterogeneity of β-cells in terms of the time-varying β-cell formation and loss estimated by a β-cell turnover model. This modeling approach was applied to evaluate β-cell lifespan in a rodent model of type 2 diabetes in comparison with nondiabetic controls. When rats were 10 mo old, mean β-cell lifespan was 1 mo vs. 6 mo in rats with type 2 diabetes vs. controls. A shortened β-cell lifespan in a rat model of type 2 diabetes results in a decrease in mean β-cell age and thus contributes to decreased β-cell mass.     

A Serine Protease in Soybean Seeds That Acts Specifically on the Native {alpha} Subunit of {beta}-Conglycinin

A proteolytic activity directed against the     

Effect of light treatment and endogenous growth hormones on {alpha}-and {beta}-amylase activities in cotyledons of Phaseolus vulgaris L.

The influence of light and endogenous plant-growth regulatorson the evolution of - and ß-amylases in cotyledonsof Phaseolus vulgaris L. was investigated. Both enzymes, whichare not present in ungerminated seeds, appear during germinationof intact seedlings or incubation of excised cotyledons. -Amylaseactivity decreases upon exposure to light. This inhibition iscorrelated with a drastic increase in chlorophyll content anda change in the endogenous gibberellin-inhibitor balance. ß-Amylaseactivity was not affected by light treatment but was by thepresence of endogenous cytokinins. (Received February 3, 1977; )     

Developmental Variation of the Neurotoxin, {beta}-N-Oxalyl-L-{alpha},{beta}-diamino propionic acid (ODAP), in Lathyrus sativus

Several species of the tribe Viceae (Leguminosae) produce non-proteinamino acids that are toxic to man and animals. The neurotoxinß-N-oxalyl-L-,ß-diamino propionic acid (ODAP)in cultivated Lathyrus sativus causes human neurolathyrism,a neurological disease resulting in the paralysis of lower limbs.Surveys have shown that there are large scale variations betweenspecies of Lathyrus and varieties of L. sativus for the amountof cellular ODAP. In the present investigation, thin-layer chromatographyand chemical analysis were used to study developmental variationin the amount of ODAP in tissues and organs of L. sativus. Theresults confirmed that the rate of synthesis and accumulationof ODAP varied during plant development. Increased rates ofsynthesis were confirmed in young seedlings and in the developingfruits of L. sativus.Copyright 1994, 1999 Academic Press Lathyrus sativus, neurotoxin, ODAP, plant development     

Activation-dependent conformational changes in {beta}-arrestin 2

Beta-arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and beta-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in beta-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in beta-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of beta-arrestin 2 in the presence of a phosphopeptide (V(2)R-pp) derived from the C terminus of the vasopressin type II receptor (V(2)R) or the corresponding nonphosphopeptide (V(2)R-np). V(2)R-pp binds to beta-arrestin 2 specifically, whereas V(2)R-np does not. Activation of beta-arrestin 2 upon V(2)R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of beta-arrestin 2; however, heparin and V(2)R-pp have different binding site(s) and/or induce different conformational changes in beta-arrestin 2. Release of the C terminus from the rest of beta-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to beta-arrestin 2 by 10-fold. Thus, the V(2)R-pp can activate beta-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of beta-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-beta-arrestin interactions can be modeled in vitro.     

Incorporation of r{beta}-hydroxy-r{beta}-methylglutaric acid into carotenoids and other ether soluble compounds in maize seedlings

3-¹⁴C-rß-hydroxy-rß-methylglutaric acid(HMG) was effectively incorporated into isoprenoids by excisedetiolatcd shoots as well as by the cell-free extracts of maize.The rate of incorporation indicated that HMG was not degradedto acetate or acetoacetate before entering the isoprenoid pathway.HMG and HMG-CoA were equally incorporated by the soluble extractinto carotenoids indicating that, in addition to HMG-CoA reductase(EC.1.1.1.34), HMG activating enzyme was also present in theplant. The soluble system (20,000 x g fraction) showed a pHoptimum of 7. Endogenous metabolites such as mevalonic acid(MVA) in the reaction mixture decreased the incorporation ofHMG into isoprenoids. (Received September 21, 1971; )     

A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import

Importin-beta (Impbeta) is a major transport receptor for Ran-dependent import of nuclear cargo. Impbeta can bind cargo directly or through an adaptor such as Importin-alpha (Impalpha). Factors involved in nuclear transport have been well studied, but systems analysis can offer further insight into regulatory mechanisms. We used computer simulation and real-time assays in intact cells to examine Impalpha-beta-mediated import. The model reflects experimentally determined rates for cargo import and correctly predicts that import is limited principally by Impalpha and Ran, but is also sensitive to NTF2. The model predicts that CAS is not limiting for the initial rate of cargo import and, surprisingly, that increased concentrations of Impbeta and the exchange factor, RCC1, actually inhibit rather than stimulate import. These unexpected predictions were all validated experimentally. The model revealed that inhibition by RCC1 is caused by sequestration of nuclear Ran. Inhibition by Impbeta results from depletion nuclear RanGTP, and, in support of this mechanism, expression of mRFP-Ran reversed the inhibition.     

Biosynthesis of Xyloglucan in Suspension-cultured Soybean Cells. Evidence that the Enzyme System of Xyloglucan Synthesis does not Contain {beta}-1,4-Glucan 4-{beta}-D-Glucosyltransferase Activity (EC 2.4.1.12)

Xyloglucan 4-ß-D-glucosyltransferase, an enzyme responsiblefor the formation of the xyloglucan backbone, in a particulatepreparation of soybean cells has been compared with ß-1,4-glucan4-ß-D-glucosyltransferase of the same origin. Thefollowing observations indicate that the enzyme system of xyloglucansynthesis does not contain ß-1,4-glucan 4-ß-D-glucosyltransferaseactivity, although both enzymes transfer the glucosyl residuefrom UDP-glucose to form the ß-1,4-glucosidic linkage:1. The incorporation of [¹⁴C]glucose into xyloglucan dependedon the presence of UDP-xylose in the incubation mixture. 2.No measurable amount of radioactivity was incorporated fromUDP-[¹⁴C]xylose into the cello-oligosaccharides, although theincorporation of [¹⁴C]xylose into xyloglucan depended on thepresence of UDP-glucose in the incubation mixture (Hayashi andMatsuda 1981b). 3. The activity of xyloglucan 4-ß-D-glucosyltransferasewas stimulated more strongly by Mn²⁺ than by Mg²⁺, whereas Mg²⁺was the most active stimulator for the activity of ß-1,4-glucan4-ß-D-glucosyltransferase. 4. An addition of GDP-glucose(100 µM) to the incubation mixture inhibited the activityof xyloglucan 4-ß-D-glucosyltransferase by 17%, whereasthe activity of ß-1,4-glucan 4-ß-D-glucosyltransferasewas inhibited 56% under the same conditions. 5. Irpex exo-cellulasedid not hydrolyze the xyloglucan synthesized in vitro. 6. Theß-1,4-glucan synthesized in vitro was not a branchedxyloglucan because it gave no 2,3-di-O-methyl glucose derivativeon methylation analysis. 7. Pulse-chase experiments indicatedthat the ß-1,4-glucan was not transformed into thexyloglucan. The subcellular distribution of the xyloglucan synthase, however,was similar to that of the ß-1,4-glucan synthase (Golgi-located1,4-ß-D-glucan 4-ß-D-glucosyltransferase).Thus, it appears that the latter enzyme is located at a siteclose to xyloglucan synthase and is set aside for the assemblyof these polysaccharides into the plant cell surface. (Received May 21, 1981; Accepted October 13, 1981)     

Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems

In the present study we have optimized the concentration of the bleaching herbicide norflurazon to obtain Dunaliella bardawil cells able to accumulate phytoene without losing viability. The highest concentration of phytoene 10.4 g/gChl was obtained for a concentration of norflurazon of 10 microg/mL. Norflurazon-treated Dunaliella bardawil cells are able to accumulate high concentrations of phytoene if the carotenogenic pathway is stimulated, but the lack of colored carotenoids make these cells particularly sensitive to high light intensities and to UVB radiation, so other stimuli, such as nitrogen starvation, have to be used to force the accumulation of phytoene. Detailed time-course evolution of the carotenoids lutein, violaxanthin, zeaxanthin, phytene and beta-carotene and the photosynthetic pigment chlorophyll was followed upon transfer of Dunaliella bardawil cells to nitrogen starvation in presence and absence of norflurazon. The combined use of the carotenogenic pathway inhibitor norflurazon and biphasic aqueous/organic systems to force the excretion of phytoene into the culture medium has been investigated. Cells cultured in the biphasic system were viable and able to produce phytoene during 3 days. Futhermore the productivity increased from 0.14 g/gChl . h in the aqueous culture to 0.18 g/gChl . h in the biphasic system. About 15% of the total phytoene produced by Dunaliella bardawil was excreted and immediately partionated into the organic phase. The concentration of phytoene in the decane phase was 2.05 g/gChl after 72 h, this means that about 47 g of phytoene per litre of culture were in the organic phase.     

Transcription of the {beta}-galactoside {alpha}2,6-sialyltransferase gene in B lymphocytes is directed by a separate and distinct promoter{dagger}

A single human gene, SIAT1, encodes the ß-galactoside     

A novel {beta}-galactosidase gene isolated from the bacterium Xanthomonas manihotis exhibits strong homology to several eukaryotic {beta}-galactosidases

The gene encoding a ß-galactosidase from Xanthomonasmanihotis was cloned into Escherichia coli. The gene resideson a 2.4 kb DNA fragment which was isolated from a partial Sau3Alibrary in the cloning vector pUC19 using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as the selection. The enzyme produced by the clone hasa specificity for ß1-3->ß1-4-linked galactose.The nucleotide sequence of the gene was determined. The deducedprotein sequence contained 597 amino acids yielding a monomericmolecular mass of 66 kDa. The cloned ß-galactosidaseshowed no similarity to any known prokaryotic ß-galactosidase.However, extensive similarity was observed with eukaryotic ß-galactosidasesfrom animals, plants and fungi. The strongest similarity waswith the ß-galactosidases found hi the human and mouselysosomes (42 and 41% identity, respectively). Alignment ofthe X.manihotis and eukaryotic ß-galactosidase sequencesrevealed seven highly conserved domains common to each protein.Additionally, Domain 1 in X.manihotis showed similarity to regionswithin catalytic domains from seven xylanases and cellulasesbelonging to family 10 of glucosyl hydrolases. A region spanningDomain 2 showed similarity to the catalytic domain of endo ß1-3glucanases from tobacco and barley. cellulase ß-galactosidase G_M$$$gangliosidosis Morquio B syndrome Xanthomonas     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)