Cre-mediated seed-specific transgene excision in tobacco.

Here we report the production of marker-free transgenic plants expressing phenolic compounds with high pharmacological value. Our strategy consisted in simultaneous delivery of lox-target and cre-containing constructs into the plant genome by cotransformation. In the Cre-vector, the cre recombinase gene was controlled by a seed-specific napin promoter. In the lox-target construct the selectable bar gene was placed between two lox sites in direct orientation, while a napin promoter driven vstI gene was inserted outside of the lox sites. Upon seed-specific cre induction the bar expression cassette was excised from the tobacco genome. Genetic and molecular analysis of T1 progeny plants indicated DNA excision in all 10 transgenic lines tested. RP-HPLC analysis demonstrated that the expression of the vstI gene resulted in accumulation of trans-resveratrol and its glycosylated derivative piceid in seeds of all marker free lines. These findings indicate that the seed-specific marker gene excision did not interfere with the expression of the gene of interest. Our data demonstrated the feasi of a developmentally controlled cre gene to mediate site-specific excision in tobacco very efficiently.

[Therapeutic effects of electrical stimulation therapy on vocal fold vibration irregularity]. / Therapeutische Beeinflussung von Schwingungs-irregularitäten durch Elektrostimulationstherapie.

BACKGROUND: Vocal fold vibration may be adversely affected by numerous conditions. We studied whether electrical stimulation can alleviate vocal fold vibration irregularity. METHODS: A total of 90 patients with varying degrees of vocal fold vibration irregularity due to unilateral vocal fold paresis were recruited and received either electrical stimulation therapy or a voice exercise/behavioral treatment. Vocal fold vibration irregularity was calculated from a speech sample before and after therapy. RESULTS: After 3 months, the increase in vibration stability was significantly greater for patients who received electrical stimulation therapy compared with patients who received traditional voice therapy. DISCUSSION: Voice control includes vocal fold vibration regularity. It appears that electrical stimulation therapy can be used effectively in patients with vocal fold paresis and concomitant loss of voice control.

Hydroxycinnamoyltransferases Involved in the Accumulation of Caffeic Acid Esters in Gametophytes and Sporophytes of Equisetum arvense.

Four hydroxycinnamoyltransferases from Equisetum arvense L. were studied that catalyze the formation of mono-O-caffeoyl-meso-tartrate, di-O-caffeoyl-meso-tartrate, 5-O-caffeoylshikimate (dactylifrate), and 5-O-caffeoylquinate (chlorogenate). The enzymes were classified as coenzyme A (CoA)-ester-dependent acyltransferases (EC 2.3.1), i.e. hydroxycinnamoyl-CoA:meso-tartrate hydroxycinnamoyltransferase (CTT), hydroxycinnamoyl-CoA:caf-feoyl-meso-tartrate hydroxycinnamoyltransferase (CCT), hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CST), and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase. The CTT, CCT, and CST were partially purified and separated from E. arvense gametophytes by hydrophobic interaction chromatography on Fractogel TSK Butyl-650 followed by molecular exclusion on fast protein liquid chromatography-Superdex-75 with 87-, 62-, and 130- fold enrichments and 12, 8, and 11% yields, respectively. The enzyme activities obtained with caffeoyl-CoA were 95 (CTT), 74 (CCT), and 200 [mu]kat (CST) kg-1 protein. The apparent native relative molecular weight values were found to be approximately 45,000 (CTT), 52,000 (CCT), and 50,000 (CST). Each enzyme showed highest activities at pH 7.5, the CCT and CST in Tris-HCl (1.2 and 1.0 M) and the CTT in imidazole-HCl (1.25 M). Enzyme activities were stimulated more than 3-fold by 100 mM ascorbate. The apparent energies of activation (kilojoules mol-1) were calculated to be 56 (CTT), 69 (CST), and 76 (CCT). The enzymes accepted cinnamoyl-CoA and various hydroxycinnamoyl-CoAs. The time course of the transferase activities along with that of a fourth one, hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, and the pattern of product accumulation were determined during a 1-year growth period of the E. arvense sporophytes.

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum.

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris "Golden Beet") showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris "Altamo") plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.

Tissue-Specific and Development-Dependent Accumulation of Phenylpropanoids in Larch Mycorrhizas.

The tissue-specific and development-dependent accumulation of secondary products in roots and mycorrhizas of larch (Larix decidua Mill.; Pinaceae) was studied using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble catechin, epicatechin, quercetin 3-O-[alpha]-rhamnoside, cyanidin- and peonidin 3-O-[beta]-glucoside, 4-O-[beta]-hydroxybenzoyl-O-[beta]-glucose, 4-hydroxybenzoate 4-O-[beta]-glucoside, maltol 3-O-[beta]-glucoside, and the wall-bound 4-hydroxybenzaldehyde, vanillin, and ferulate. In addition, we partially identified a tetrahydroxystilbene monoglycoside, a quercetin glycoside, and eight oligomeric proanthocyanidins. Comparison between the compounds accumulating in the apical tissue of fine roots, long roots, and in vitro grown mycorrhizas (L. decidua-Suillus tridentinus) showed elevated levels of the major compounds catechin and epicatechin as well as the minor compound 4-hydroxybenzoate 4-O-[beta]-glucoside specifically in the root apex of young mycorrhizas. The amounts of wall-bound 4-hydroxybenzaldehyde and vanillin were increased in all of the mycorrhizal sections examined. During the early stages of mycorrhization the concentrations of these compounds increased rapidly, perhaps induced by the mycorrhizal fungus. In addition, studies of L. decidua-Boletinus cavipes mycorrhizas from a natural stand showed that the central part of the subapical cortex tissue and the endodermis both accumulate massive concentrations of catechin, epicatechin, and wall-bound ferulate compared with the outer part of the cortex, where the Hartig net is being formed.

Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase.

Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47-52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.

Secondary products in mycorrhizal roots of tobacco and tomato.

Colonization of the roots of various tobacco species and cultivars (Nicotiana glauca Grah., N. longiflora Cav., N. rustica L., N. tabacum L., N. tabacum L. cv. Samsun NN, N. sanderae hort. Sander ex Wats.) as well as tomato plants (Lycopersicon esculentum L. cv. Moneymaker) by the arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith resulted in the accumulation of several glycosylated C13 cyclohexenone derivatives. Eight derivatives were isolated from the mycorrhizal roots by preparative high performance liquid chromatography (HPLC) and spectroscopically identified (MS and NMR) as mono-, di- and triglucosides of 6-(9-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one and monoglucosides of 6-(9-hydroxybutyl)-1,5-dimethyl-4-cyclohexen-3-one-1-carboxylic acid and 6-(9-hydroxybutyl)-1,1-dimethyl-4-cyclohexen-3-one-5-carboxylic acid. In contrast to the induced cyclohexenone derivatives, accumulation of the coumarins scopoletin and its glucoside (scopolin) in roots of N. glauca Grah. and N. tabacum L. cv. Samsun NN, was markedly suppressed.

Betacyanins from bracts of Bougainvillea glabra.

Betacyanins from the bracts of Bougainvillea glabra were isolated and characterized by a combination of spectroscopic techniques (DAD-HPLC, NMR, LC-MS, GC-MS, electrospray MS, tandem MS) as gomphrenin I (betanidin 6-O-beta-glucoside) and various derivatives of bougainvillein-v (betanidin 6-O-beta-sophoroside), i.e. mono- and diglucosylsophorosides which are acylated with 4-coumaric and caffeic acid (mono- and diesters). Besides the betacyanins, B. glabra bracts accumulated large amounts of flavonols (kaempferol and quercetin conjugates) reaching ratios of flavonol to betacyanin of 1:1.

Anthocyanins from cell suspension cultures of Daucus carota.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2''-O-beta-D-xylopyranosyl-6''-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.

Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase.

The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were analyzed. Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants). The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e. N-caffeoylputrescine. In addition, the level of the coumarin glucoside scopolin was markedly enhanced. Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens. This is consistent with results from potato virus Y-infected plants. Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.

Betacyanins from plants and cell cultures of Phytolacca americana.

Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

Light-induced betacyanin and flavonol accumulation in bladder cells of Mesembryanthemum crystallinum.

Treatment of the halophyte Mesembryanthemum crystallinum L. (ice plant) (Aizoaceae) with high intensities of white light resulted in a rapid cell-specific accumulation of betacyanins and flavonoids with 6-methoxyisorhamnetin 3-O-¿[(2"'-E-feruloyl)-3"'-O-(beta-D- glucopyranosyl)](2"-O-beta-D-xylopyranosyl)]-beta-D-glucopyranoside (mesembryanthin) as the predominant component, within bladder cells of the leaf epidermis. Induced accumulation of these metabolites was first detected 18 h after the initiation of light treatment in bladder cells located at the tip of young leaves followed by the bladder cells located on the epidermis of fully expanded leaves. UV-A light apparently is sufficient to induce accumulation of betacyanins and flavonoids. Application of 2-aminoindan 2-phosphonic acid, a specific inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), not only inhibited the accumulation of flavonoids but also reduced betacyanin formation. Based on these observations we suggest these bladder cells as a model system to study regulation of betacyanin and flavonoid biosyntheses.

Enzymatic Synthesis of t,2-di-Sinapoylglucose from 1-Sinapoylglucose by a Protein Preparation from Cotyledons of Raphanus sstivus Grown in the Dark.

Protein preparation from cotyledons of Raphanus sativus seedlings grown in the dark catalyzed the transfer of the sinapoyl moiety of 1-O-sinapoyl-ß-D-glucose to the C-2 hydroxyl group of the glucose moiety of another molecule of 1-O-sinapcyl-ß-D-glucose to form 1,2-di-O-sinapoyl-ß-D-glucopyranose. In this synthesis the 1-O-acyl glucoside was used both as acyl donor and acceptor molecule. Maximal rate of product formation was found to be at pH 7.5 in 0.1M Hepes buffer.

Blood volume of nonsplenectomized and splenectomized cats before and after acute hemorrhage.

Blood volume (BV) was determined in awake, nonsplenectomized (NSPX) and splenectomized (SPX) cats before and after hemorrhage (6 ml/kg). Each NSPX cat had a determined BV at least 10 ml/kg greater than the same cat after splenectomy. The mean BV of SPX cats was 43.4 +/- 8.94. ml kg (4.3% of body weight). The calculated RBC masses of NSPX and SPX cats were 17.0 +/- 4.07 and 12.2 +/- 1.12 ml/kg, respectively. Each NSPX cat had apparent RBC masses of 5 ml/kg greater than that of the same cat after splenectomy was done. At 1 hour after a hemorrhage, the BV and RBC masses determined in SPX cats were 46.7 +/- 12.1 and 9.7 +/- 1.90 ml/kg, respectively. Extravascular-to-intravascular fluid flux (calculated from RBC masses and plasma protein dilution) was approximately 0.80% of body weight. The indirect method with 51Cr-labeled RBC for BV determination was accurate and precise in awake, SPX cats; in awake, NSPX cats, the 51Cr-labeled RBC dilution method was precise, but not accurate. The spleen in the cat resulted in marked overestimations of BV and RBC masses.

Effects of the spleen, epinephrine, and splenectomy on determination of blood volume in cats.

Blood volume (BV), RBC mass, plasma protein (PP), and PCV were determined in (i) awake, nonsplenectomized (NSPX) cats before and after splenic contraction was induced by IV administration of epinephrine, (ii) in pentobarbital-anesthetized, NSPX cats before and after splenic contraction was induced by epinephrine and splenectomy, and (iii) in awake, splenectomized (SPX) cats before and after epinephrine was administered. Blood volume, absolute RBC mass in the spleen could not be determined accurately in awake or anesthetized, NSPX cats. Accurate BV and RBC mass determinations could only be obtained in cats without the spleen; mean BV and RBC mass of awake or anesthetized, SPX cats were 4.1% and 1.4% or body weight, respectively. The spleen was a dynamic sequestering organ for feline erythrocytes (RBC). Epinephrine (0.1 ml of 1:1,000) given IV resulted in the observed contraction of the spleen within 2 minutes. Relaxation of the spleen with RBC uptake began 3 minutes after epinephrine was given and it proceeded at a rate of approximately 0.93% of circulating PCV/min. At 20 minutes after epinephrine was given, a steady state of PCV occurred, and 20% of the maximum circulating PCV observed after the administration of epinephrine was stored within the spleen. At 60 minutes after epinephrine was given to awake, SPX cats, intravascular fluid influx of 4 ml/kg resulted in a decrease of PCV and PP. At 3 hours after splenectomy was done, intravascular fluid efflux resulted in hemoconcentration--a calculated fluid deficit of 2.5 ml/kg/hr occurring during the 3 hours after surgical removal of the spleen.

alpha-Adrenoreceptor stimulation of the systemic circulation in dogs.

The influence of phenylephrine (an alpha-adrenoreceptor agonist) on the arterial and venous systems of the systemic circulation was studied in 10 anesthetized dogs during a right ventricular bypass procedure. Phenylephrine (1 microgram X kg-1 of body weight X min-1) produced a significant (P less than 0.001) increase in arterial resistance, no change in venous resistance, and a mild decrease in venous compliance. Seemingly in the clinically normal dog, a dose of phenylephrine sufficient to double total peripheral resistance will redistribute a small amount of blood volume from the venous to arterial systems with a slight decrease in venous return and concomitant attenuation of cardiac output.

Valved apico-aortic conduit for relief of left ventricular hypertension caused by discrete subaortic stenosis in dogs.

A double-outlet left ventricle (LV), with a valved conduit interposed between the LV apex and the aorta, was created in 7 dogs with subaortic stenosis. Of 8 dogs in which the implantation was attempted, 1 died following thoracotomy but before conduit implantation could be performed, 1 died from hemorrhage 24 hours after surgery, 1 died from septicemia as a sequel to pneumonia 10 days after surgery, 1 died from "shock-lung" 4 days after surgery, and 4 were functionally normal 22, 12, 6, and 2 months after surgery. In the 7 dogs in which the implantation was completed, the mean LV to aorta (LV-Ao) pressure gradient was significantly (P less than 0.01) reduced by the implantation. Diastolic and systolic murmurs were detected over the prosthetic valve area in 3 of the 4 surviving dogs 1 to 4 days postoperatively, but the murmurs gradually decreased in intensity until they disappeared after 1 month. The 4 survivors had no angiographic evidence of prosthetic valve insufficiency at 2 months or at 1 year. In 3 of the survivors, the LV-Ao pressure gradients 2 months postoperatively were 45, 20, and 0 mm of Hg, as compared with 120, 90, and 50 mm of Hg preoperatively. Postoperative pressure measurements were not obtained on 1 surviving dog.