Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Characterization of an enantioselective amidase with potential application to asymmetric hydrolysis of (<Emphasis Type="Italic">R</Emphasis>, <Emphasis Type="Italic">S</Emphasis>)-2, 2-dimethylcyclopropane carboxamide

Amidase is a promising synthesis tool for chiral amides and related derivatives. In the present study, the biochemical properties of the Delftia tsuruhatensis CCTCC M 205114 enantioselective amidase were determined for its potential application in chiral amides synthesis. D. tsuruhatensis CCTCC M 205114 amidase was purified 105.2 fold with total activity recovery of 4.26%. The enzyme is a monomer with a subunit of approximately 50 kDa by analytical gel filtration HPLC and SDS–PAGE. It had a broad substrate spectrum and displayed high enantioselectivity against R-2, 2-dimethylcyclopropane carboxamide and R-mandelic amide. The amidase was applied to enantioselective hydrolysis of R-2, 2-dimethylcyclopropane carboxamide from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate S-2, 2-dimethylcyclopropane carboxamide. This enzyme did not require metal ions for the hydrolysis reaction. Its optimal pH and temperature were 8.0 and 35°C, respectively. The K _m and V _max of the amidase for R-2, 2-dimethylcyclopropane carboxamide were 2.54 mM and 8.37 μmol min⁻¹ mg protein⁻¹, respectively. After 60 min of the reaction, R-2, 2-dimethylcyclopropane carboxamide was completely hydrolyzed, generating S-2, 2-dimethylcyclopropane carboxamide with a yield of 45.9% and an e.e. of above 99%. Therefore, this amidase can serve as a promising producer for S-2, 2-dimethylcyclopropane carboxamide and other amides.     

Numerical Taxonomy of <Emphasis Type="Italic">Vibrionaceae</Emphasis> Isolated from Cultured Amberjack (<Emphasis Type="Italic">Seriola dumerili</Emphasis>) and Surrounding Water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S_SM and S_J coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S_SM values ≥ 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish. Received: 27 March 2002 / Accepted: 24 May 2002     

Synthesis of (<Emphasis Type="Italic">2S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4-phenylamino-5-oxoproline derivatives

Summary. The paper describes the synthesis of (2S,4S)-4-(N-Ts)- and (2S,4S)-4-(N-Boc)-phenylamino-5-oxoprolines (pyroglutamic acid). These derivatives have been shown to be useful for synthesis of their amides and peptides in spite of steric hindrances caused by bulky groups adjacent to the reaction centre. Under the conditions applied no lactam ring opening and no loss of stereochemical integrity of any of the chiral centres were observed, which has been confirmed by NMR techniques. Received December 29, 2000 Accepted June 26, 2001     

Seasonal and regional occurrence of <Emphasis Type="Italic">Acanthocephalus</Emphasis> sp. (Acanthocephala: Echinorhynchidae) in fishes and isopods (<Emphasis Type="Italic">Asellus hilgendorfi</Emphasis>) in a lake system in northern Japan

 The echinorhynchid acanthocephalan Acanthocephalus sp. was collected and described from four species of fishes (rainbow trout Oncorhynchus mykiss, Sakhalin huchen Hucho perryi, Japanese pond smelt Hypomesus nipponensis, and threespine stickleback Gasterosteus aculeatus) from a lake system, the Tsugaru-Jūniko Lakes, in Aomori Prefecture, northern Japan. In rainbow trout, the prevalence and intensity of infection markedly differed between lakes, and the fish were most frequently and most heavily infected in the lakes with a dense population of the isopod intermediate host Asellus hilgendorfi. In isopods, the prevalence of acanthocephalan larvae increased in the late winter and reached its highest level in March or April. In rainbow trout, male worms were abundant from winter to spring, and female worms were immature during these seasons. Gravid females were abundant in summer and autumn. These findings indicate that Acanthocephalus sp. is an annual species and its recruitment from the intermediate host to the fish occurs mainly in winter and spring. Received: January 9, 2002 / Accepted: April 18, 2002 Acknowledgments We thank Professor Shōichi Saito, Faculty of Education, Hirosaki University, for his encouragement during this study. Thanks are also due to many students of the Nature Study Laboratory, Faculty of Education, Hirosaki University, for their assistance in the field. We are greatly indebted to the Iwasaki Village Office and Fukaura Forestry Office for giving us permission for the survey. Correspondence to:A. Ohtaka     

Chlamydospore formation of <Emphasis Type="Italic">Entoloma clypeatum</Emphasis> f. <Emphasis Type="Italic">hybridum </Emphasis>on mycorrhizas and rhizomorphs associated with <Emphasis Type="Italic">Rosa multiflora</Emphasis>

 Chlamydospores of Entoloma clypeatum f. hybridum were described on the mycorrhizas and rhizomorphs associated with Rosa multiflora. Their developmental pattern seems to be the Nyctalis type. This is the first report on chlamydospore formation on the mycorrhizae in entolomatoid fungi. Received: January 17, 2002 / Accepted: November 5, 2002 Acknowledgments K.H. is grateful to Emeritus Professor N. Sagara in Kyoto University, in whose laboratory part of this study was undertaken. Thanks are due to Mr. D. Sakuma for allowing the specimens to be kept in Osaka Museum of Natural History. Correspondence to:H. Kobayashi     

Effects of <Emphasis Type="Italic">Acanthocephalus</Emphasis> sp. (Acanthocephala: Echinorhynchidae) on the body size and reproduction of isopods (<Emphasis Type="Italic">Asellus hilgendorfi</Emphasis>)

 Isopods Asellus hilgendorfi were collected from a small lake in northern Japan and examined to determine whether their body size and reproduction were affected by infection with larval acanthocephalans (Acanthocephalus sp.). Seasonal changes in the breeding ratio of isopods and the prevalence of larval acanthocephalan infection showed a reverse trend. Acanthocephalan larvae occurred mainly in males and immature females and were rarely found in mature females. In late immature females, the body size, as indicated by the width of the pleotelson, of infected isopods was significantly larger than that of uninfected ones. These results suggest that acanthocephalans can prevent female isopods from attaining sexual maturity and increasing their body size. Received: January 9, 2002 / Accepted: December 16, 2002 Acknowledgments We thank Professor Shōichi Saito, Faculty of Education, Hirosaki University, for his encouragement of the present study. Thanks are also due to the Iwasaki Village Office and the Fukaura Forestry Office for giving us permission for the survey. Correspondence to:A. Ohtaka     

Facile synthesis of optically pure (<Emphasis Type="Italic">S</Emphasis>)-3-<Emphasis Type="Italic">p</Emphasis>-hydroxyphenyllactic acid derivatives

Summary. Optically pure (S)-3-p-hydroxyphenyllactic acid derivatives are important intermediates of peroxisome proliferator-activated receptor α/γ dual agonists and heteropeptides. Many efforts have been made for synthesis of those intermediates, but there exist some flaws yet. We observed that dielectric constants of organic solvents drastically affected diazotization of O-benzyl-L-tyrosine. Optically pure (S)-3-p-benzyloxyphenyllactic acid was obtained by simple recrystallization when DMF or DMSO of higher dielectric constant was used as a co-solvent in diazotization of O-benzyl-L-tyrosine. It was easily turned into various optically pure (S)-3-p-hydroxyphenyllactic acid derivatives.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria <Emphasis Type="Italic">Mycobacterium neoaurum,Pimelobacter simplex</Emphasis>, and <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.     

Protein cysteine <Emphasis Type="Italic">S</Emphasis>-guanylation and electrophilic signal transduction by endogenous nitro-nucleotides

Nitric oxide (NO), a gaseous free radical that is synthesized in organisms by nitric oxide synthases, participates in a critical fashion in the regulation of diverse physiological functions such as vascular and neuronal signal transduction, host defense, and cell death regulation. Two major pathways of NO signaling involve production of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and posttranslational modification (PTM) of redox-sensitive cysteine thiols of proteins. We recently clarified the physiological formation of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) as the first demonstration, since the discovery of cGMP more than 40 years ago, of a new second messenger derived from cGMP in mammals. 8-Nitro-cGMP is electrophilic and reacts efficiently with sulfhydryls of proteins to produce a novel PTM via cGMP adduction, a process that we named protein S-guanylation. 8-Nitro-cGMP may regulate electrophilic signaling on the basis of its electrophilicity through induction of S-guanylation of redox sensor proteins. Examples include S-guanylation of the redox sensor protein Kelch-like ECH-associated protein 1 (Keap1), which leads to activation of NF-E2-related factor 2 (Nrf2)-dependent expression of antioxidant and cytoprotective genes. This S-guanylation-mediated activation of an antioxidant adaptive response may play an important role in cytoprotection during bacterial infections and oxidative stress. Identification of new redox-sensitive proteins as targets for S-guanylation may help development of novel therapeutics for oxidative stress- and inflammation-related disorders and vascular diseases as well as understanding of cellular protection against oxidative stress.     

Comparison of the Antioxidant Properties and the Toxicity of <Emphasis Type="Italic">p</Emphasis>,<Emphasis Type="Italic">p</Emphasis>′-Dichlorodiphenyl Ditelluride with the Parent Compound,Diphenyl Ditelluride

The hypothesis to be tested in this study is whether the introduction of the chloro group into diphenyl ditelluride molecule (p,p′-dichlorodiphenyl ditelluride, compound 1b) alters the antioxidant and scavenging activity of diphenyl ditelluride (compound 1a) in vitro. The results revealed that 1a and 1b had a potent antioxidant activity in vitro. However, the introduction of a functional group, chloro, into diphenyl ditelluride molecule (1b) did not cause great alterations in the antioxidant action of diphenyl ditelluride against lipid peroxidation, protein carbonyl, and scavenging of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) and 2,2′-diphenyl-1-picrylhydrazyl radicals. Based on the in vitro results, different doses (0.25 and 0.75 μmol/kg) of 1a and 1b or vehicle (canola oil, 1 ml/kg) were administered to rats to investigate if the presence of chloro into diphenyl ditelluride molecule reduces its toxicity. The data demonstrate that the chloro group introduced into diphenyl ditelluride molecule did not alter the acute oral toxicity in rats. The administration of compound 1a in rats only altered the urea level, while compound 1b caused alterations in all toxicological parameters analyzed (alanine aminotransferase and aspartate aminotransferase activities, urea and creatinine levels) in plasma of rats. The results of the present investigation support similar antioxidant and scavenging activities of 1a and 1b in rat liver homogenate in vitro. Furthermore, the presence of chloro into diphenyl ditelluride molecule did not alter the mortality index but increased toxicity of diphenyl ditelluride in rats.     

<Emphasis Type="Italic">Erysiphe patagoniaca</Emphasis>: a new species of <Emphasis Type="Italic">Erysiphe </Emphasis>sect. <Emphasis Type="Italic">Uncinula </Emphasis>from Patagonia,Argentina

 A new species of Erysiphe sect. Uncinula is described and illustrated from Patagonia, Argentina. Erysiphe patagoniaca sp. nov., found on leaves of Nothofagus × antarctica, is similar to E. nothofagi and E. kenjiana, but differs in its appendages being twisted throughout their length and the number of appendages, asci, and ascospores. The two endemic species of Erysiphe sect. Uncinula, E. magellanica and E. nothofagi, coexisted on the same leaves together with Erysiphe patagoniaca. Received: September 19, 2002 / Accepted: November 28, 2002 Acknowledgments The authors are grateful to Ms. Seiko Niinomi for providing the micrographs of ascomata of Erysiphe spp. on Nothofagus. Correspondence to:S. Takamatsu     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

Molecular characterization,recombinant expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and biological activity of (<Emphasis Type="Italic">S</Emphasis>)-Tetrahydroberberine oxidase from <Emphasis Type="Italic">Corydalis saxicola</Emphasis> Bunt

(S)-Tetrahydroberberine [(S)-THB] oxidase is the last enzyme of benzylisoquinoline alkaloids pathway which catalyzes the dehydrogenation of four hydrogen atoms of (S)-THB to produce berberine, the final step of berberine biosynthesis. A (S)-THB gene, designated as Cs(S)-THBO (Genbank accession No. HQ393909), was cloned from a Corydalis saxicola cDNA library by rapid amplification of cDNA ends. The full-length of cDNA of Cs(S)-THBO was 1127 bp with an open reading frame of 699 bp that predicted to encode a 232-amino acid polypeptide, with a predicted molecular mass of 25.20 kDa. Cs(S)-THBO was the first (S)-THBO gene found in C. saxicola. Real-time quantitative PCR analysis indicated that Cs(S)-THBO was constitutively expressed in roots, stems, leaves and flowers of C. saxicola, and with the highest expression level in roots. The results of treatment experiment for plant defense responses revealed that expression of Cs(S)-THBO had a prominent diversity. Recombinant Cs(S)-THBO protein expressed in Escherichia coli strain BL21 (DE3) was active. The results of feeding experiment and HPLC–DAD–ESI–MSⁿ analysis showed that Cs(S)-THBO had the function of catalyzing (S)-tetrahydroberberine to berberine.     

Co-expression of<Emphasis Type="Italic">flavonoid 3′ 5′-hydroxylase</Emphasis> and<Emphasis Type="Italic">flavonoid 3′-hydroxylase</Emphasis> Accelerates Decolorization in Transgenic Chrysanthemum Petals

Theflavonoid 3′,5′-hydroxylase (F3′,5′H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation. Cotyledon expiants were co-cultured withA. tumefaciens LBA 4404 harboring the vector pMBP that carriesF3′,5′H under the control of the CaMV 35S promoter andnptll as a selectable marker gene. After 72 h of co-cultivation, the expiants were placed on an MS medium supplemented with 4 mg L^-1 BA, 0.1 mg L^-1 NAA, 400 mg L^-1 carbenicillin, and 100 mg L^-1; kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were then rooted and acclimatized in potting soil. Integration ofF3′,5′H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed thatF3′,5′H andflavonoid 3′hydroxylase (F3′H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color results from 1) competition between levels of transgenicF3′,5′H and endogenousF3′H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of chrysanthemumDFR (dihydroflavonol 4-reductase).     

<Emphasis Type="Italic">Umbelopsis gibberispora</Emphasis> sp. nov. from Japanese leaf litter and a clarification of <Emphasis Type="Italic">Micromucor ramannianus </Emphasis>var. <Emphasis Type="Italic">angulisporus</Emphasis>

 Umbelopsis gibberispora is described as a new species in the genus Umbelopsis, Umbelopsidaceae, Mucorales. The species differs from others in this genus by ellipsoidal sporangiospores with unilaterally thickened walls. Phylogenetic analyses based on nuclear large subunit ribosomal DNA (nLSU rDNA) partial sequences suggest that U. gibberispora, U. swartii, and U. westeae form a clade together with the strains of Umbelopsis ramanniana. The ex-type strain of Micromucor ramannianus var. angulisporus is found to be very close to Umbelopsis vinacea, whereas other isolates identified under the former name in the sense of Linnemann fall in the U. ramanniana subclade. For these isolates, a new species, Umbelopsis angularis, is introduced. Phylogenetic relationships among Umbelopsis species are discussed related to their attributes of the sporangial wall and mature spore shapes. Received: August 27, 2002 / Accepted: March 11, 2003 Acknowledgments We thank Dr. Takashi Ohsono, Graduate School of Agriculture, Kyoto University, Japan, for providing the strain of U. gibberispora (CBS 109328). We also thank Dr. Wieland Meyer, University of Sydney, Australia for access to the phylogenetic tree based on ITS sequence data before publishing, and Dr. Richard C. Summerbell, Centraalbureau von Schimmelcultures, the Netherlands, for linguistic corrections.     

<Emphasis Type="Italic">Exobasidium dubium</Emphasis> and <Emphasis Type="Italic">E. miyabei</Emphasis> sp. nov. causing Exobasidium leaf blisters on <Emphasis Type="Italic">Rhododendron </Emphasis>spp. in Japan

 Two Exobasidium species causing Exobasidium leaf blister on Rhododendron spp. are described. An Exobasidium leaf blister on Rhododendron yedoense var. yedoense f. yedoense has been recognized in Hokkaido Prefecture, Japan, since the first report was issued in 1950. The causal fungus is identified with Exobasidium dubium from the morphology of its hymenial structure and mode of germination of the basidiospores. Another Exobasidium leaf blister on Rhododendron dauricum has been observed in Hokkaido Prefecture, Japan. In comparison with morphology based on hymenial structure and mode of germination of the basidiospores of the 100 validly described taxa, this fungus differs from those known taxa in the size of basidia and basidiospores, the numbers of sterigmata and septa of basidiospores, and the mode of germination of basidiospores. Thus, a new species, Exobasidium miyabei, is established and illustrated. Received: February 13, 2002 / Accepted: September 25, 2002  Present address: National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan Acknowledgments We profoundly appreciate the cooperation of Dr. V. Melnik in providing Russian papers and Dr. L. Vasilyeva for translating them into English. We thank Prof. H. Takahashi for loaning the materials in the Herbarium of the Hokkaido University Museum and Dr. W. Abe, Graduate School of Science, University of Hokkaido, for his kind help with the sampling of R. dauricum in Teshikaga, Hokkaido Prefecture. This study was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 13460019), Japan Society for the Promotion of Science (JSPS). Contribution No. 171, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Correspondence to:M. Kakishima     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.