Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis.

Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis. A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum.

[The Herz Handy--a new telemedical service concept for heart patients]. / Das Herz Handy--in neues telemedizinisches Servicekonzept für Herzpatienten.

Telemedicine is a new milestone for the health care system in the care of patients with heart disease. New technologies and the possibilities of fast data transmission have enabled this step forward. The Cardiophone offers a new telemonitoring Service Concept, which is available 24 hours a day. The patient is by the aid of the Cardiophone connected with the Medical Service Center at just the press of a button, can record and transmit an ECG and can be localized by the incorporated GPS. We report about our experiences with 363 patients over 3 years. Out of 5064 patient contacts associated with 7561 calls, 559 emergency contacts occurred. From the initial main complaints, working diagnoses were established. The final confirmed diagnoses were arrhythmias (27.8%), coronary heart disease (25.9%), psychovegetative syndrome (12.7%), backbone pain (6.2%), gastrointestinal syndrome (3.6%), others (1.8%), and exclusion diagnosis (19.9%). Ten myocardial infarctions were diagnosed, of whom 3 were confirmed; the other 7 were treated as stabile angina pectoris. Overall 823 ECG were transmitted, in average 1.6 ECGs per emergency contact. In 131 ECGs changes of the ST-segment or T-wave could be documented; 26 patients showed a pacemaker ECG. Atrial and ventricular arrhythmias as well as conduction disturbances were seen. The majority of emergency contacts (n=477, 85.3%) were successfully managed by the Service Center. In 38 emergency contacts (6.8%) admission to hospital was recommended; in 4 cases (0.7%) an ambulance was activated via the local dispatcher by the Service Center and in 29 cases (5.2%) the emergency ambulance.

CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway.

The 4- and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes. The 3-hydroxylation step leading to the formation of caffeic acid from p-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygenase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta. In this study, we show that the gene encoding CYP98A3, which was the best possible P450 candidate for a 3-hydroxylase in the Arabidopsis genome, is highly expressed in inflorescence stems and wounded tissues. Recombinant CYP98A3 expressed in yeast did not metabolize free p-coumaric acid or its glucose or CoA esters, p-coumaraldehyde, or p-coumaryl alcohol, but very actively converted the 5-O-shikimate and 5-O-d-quinate esters of trans-p-coumaric acid into the corresponding caffeic acid conjugates. The shikimate ester was converted four times faster than the quinate derivative. Antibodies directed against recombinant CYP98A3 specifically revealed differentiating vascular tissues in stem and root. Taken together, these data show that CYP98A3 catalyzes the synthesis of chlorogenic acid and very likely also the 3-hydroxylation of lignin monomers. This hydroxylation occurs on depsides, the function of which was so far not understood, revealing an additional and unexpected level of networking in lignin biosynthesis.

Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems.

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.

Cytochromes P450 for engineering herbicide tolerance.

In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.

Characterization of cis-acting elements affecting strength and phloem specificity of the coconut foliar decay virus promoter.

During replication in its host plant, coconut foliar decay virus (CFDV) remains restricted to the phloem tissue. Previous in vivo studies on subgenomic CFDV DNA had provided evidence for the phloem specificity of the CFDV promoter. Here, new promoter constructs are described which are distinguished by the presence or absence of various cis-acting signals and which gave rise to a 16-fold higher reporter gene (beta-glucuronidase) activity (reaching 30% of the cauliflower mosaic virus 35S promoter) in tobacco protoplasts, while the phloem specificity in transgenic tobacco plants was conserved. Surprisingly, the CFDV stem-loop structure dramatically influenced transcriptional efficiency. From these studies and sequence comparisons with other phloem-specific promoters, cis-signals involved in CFDV promoter strength and tissue specificity were identified.

Evidence for in vitro and in vivo autocatalytic processing of the primary translation product of beet necrotic yellow vein virus RNA 1 by a papain-like proteinase.

Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo.

[3-D echocardiography reconstruction of the thoracic aorta]. / Echokardiographische 3D-Rekonstruktion der thorakalen Aorta.

Since the beginning of the nineties the combination of echocardiography equipment with a computer algorithm for 3-D reconstruction is commercially available. The initial experiences with this unit (TomTec-system, Munich, Germany) in clinical and intraoperative settings are reported. The described unit consisted of a specially constructed transesophageal echo probe, in which the transducer (5 MHz) is mounted on a sliding carriage allowing free up and down movement in axial directions. The transducer obtains digitally dynamic 3-D information by automated pull-back of the transducer in 0.67 mm slices, gated to ECG and respiration. The information is stored on a hard disk and in the computer RAM. The acquisition time--depending on different trigger intervals--ranges from 5 to 10 minutes, usually not longer than for conventional TEE. The images are stored in special 4-D (that is 3-D in motion) data sets, allowing the calculation of any desired plane in real-time at a triggered ECG phase or dynamically. Thus this technique gives the capability of retrospective reconstruction of any view into the aorta in contrast to the omniplane TEE examination, the so-called anyplane echocardiography. In the clinical management of patients with thoracic aortic diseases transesophageal echocardiography (TEE) has become the central diagnostic tool as well as for the follow-up. This is caused by its high sensitivity and specificity comparable with that obtained by MRI. Nevertheless in a small percentage of cases there remains a questionable situation concerning the morphology of the distal ascending aorta and the aortic arch illuminated by the use of 3-D reconstruction.

The small cysteine-rich protein P14 of beet necrotic yellow vein virus regulates accumulation of RNA 2 in cis and coat protein in trans.

The effect of null mutations of the small cysteine-rich protein P14 encoded by RNA 2 of beet necrotic yellow vein virus has been investigated using in vitro transcripts of viral RNA to infect Chenopodium quinoa protoplasts. The P14 mutations down-regulated RNA 2 accumulation by approximately 10- to 50-fold. Accumulation of minus-strand RNA 2 was also diminished but RNA 1 accumulation was much less affected. The inhibition of RNA 2 accumulation could not be complemented in trans by providing P14 from another source (either a second molecule of RNA 2 or an RNA 3-based replicon) containing and expressing the P14 gene. The P14 null mutations dramatically inhibited accumulation of viral coat protein, which is encoded by the 5'-proximal gene on RNA 2, but this effect could be complemented in trans, indicating that it occurs by a mechanism distinct from that affecting RNA 2 accumulation. Transient expression experiments were also carried out in which a plasmid expressing P14 and plasmids expressing a reporter gene placed downstream of potential translational control sequences (the 5'-noncoding sequences of RNAs 2, 3, or 4) were introduced into C. quinoa or Nicotiana tabacum leaves by microprojectile bombardment. Coexpression of P14 produced a 3- to 4-fold stimulation of reporter gene expression levels for all the constructs. The lack of sequence specificity suggests that this phenomenon is not directly related to the RNA 2-specific stimulation of coat protein accumulation observed in a viral infection.

[Initial experiences with echocardiography 3D reconstruction of the thoracic aorta]. / Erste Erfahrung mit echokardiographischer 3D-Rekonstruktion der thorakalen Aorta.

Although the transesophageal echocardiography (TEE) has become the method of choice for the diagnosis as well as for the follow-up of patients with thoracic aortic aneurysms and aortic dissections, there are still some uncertainties concerning the morphology, especially that of the aorta ascendens and the aortic arch. The introduction of the 3D reconstruction now allows an exact volumetry, especially in the follow-up of aortic aneurysms, and may be helpful in better timing of the surgical intervention which is not only influenced by the horizontal diameter. Thus the 3D reconstruction of the thoracic aorta opens up a wide new field of TEE allowing a more detailed guidance preoperatively as well as a better definition of postoperative morphology.

Artificial defective interfering RNAs derived from RNA 2 of beet necrotic yellow vein virus.

Long internal deletions were introduced into cloned cDNA of beet necrotic yellow vein virus RNAs 1-4 and transcripts containing the deletions were tested for their ability to inhibit replication of viral RNA in Chenopodium quinoa protoplasts and plants. No inhibition was observed with the deletion mutants based on RNAs 1, 3 and 4 but the RNA 2 deletion mutants all provoked a dramatic inhibition of synthesis of viral RNAs 1 and 2.

Diagnosis of heart tumours by transoesophageal echocardiography: a multicentre study in 154 patients. European Cooperative Study Group.

In a retrospective multicentre study, the diagnostic potential of transoesophageal 2D-echocardiography (TEE) as compared to precordial 2D-echocardiography (TTE) was determined in 154 patients with primary or secondary tumours of the heart. Additionally, the value of standard diagnostic parameters, such as symptoms, X-ray of the chest and electrocardiogram were evaluated. In 84 patients (24 male, 60 female; age 20-85, mean 56.6 years) intracardial tumours were present, and 70 patients (37 male, 33 female; age 18-79, mean 44.3 years) presented with peri- or paracardial tumours. The main symptoms of patients with intracardial tumours were dyspnoea (60.7%), vena cava syndrome (22.2%) and chest pain (20.2%). Embolization was found in 11.9%. Left or right atrial enlargement was observed on chest X-ray in 23 patients, and echocardiographic abnormalities in 17 cases. The patients with peri- or paracardial tumours presented with dyspnoea in 51.4% of cases, loss in body weight in 20.0% and with vena cava syndrome and chest pain in 17.1%. The chest X-ray was abnormal in 56 patients. Unspecific ST segment changes in the electrocardiogram were observed in five, and arrhythmias in seven cases. Diagnosis of atrial myxomas was achieved by TTE in 95.2%, by TEE in 100%, by angiography in 78.4%, by computed tomography (CT) or magnetic resonance tomography (NMR) in 70%. Identification of the attachment point was made by angiography in 8.1%, by TTE in 64.5% and by TEE in 95.2%. In 22 patients with intracardial tumours (myxomas excepted) diagnosis was achieved by TTE in 90.9%, by TEE in 100%, by CT or NMR in 88.9% and by angiography in 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient cell-to-cell movement of beet necrotic yellow vein virus requires 3' proximal genes located on RNA 2.

RNA 2 of beet necrotic yellow vein virus (BNYVV) carries six open reading frames. The four 3' proximal frames encode the proteins P42, P13, P15, and P14. The first three species present homologies to proteins encoded by three overlapping open reading frames (the triple gene block) in potexviruses, carlaviruses, and barley stripe mosaic virus. P14 does not display homology with other known plant viral proteins. The functions of P42, P13, P15, and P14 were investigated by site-directed mutagenesis. Full-length transcripts of wild-type BNYVV RNAs 1 and 2 were infectious when coinoculated to protoplasts or leaves of Chenopodium quinoa. RNA 2 transcripts in which P42, P13, and P15 were prematurely terminated by frameshift mutations replicated in protoplasts (when inoculated with wild-type RNA 1) but were not infectious to leaves, indicating that the triple gene block proteins of BNYVV are essential for viral cell-to-cell spread. Mutations in P14 were not lethal in leaf infections but smaller local lesions and lesser amounts of viral RNA were produced. RNA 2-related subgenomic RNA species of 2.6, 1.4, and 0.7 kb were detected; they presumably direct synthesis of P42, P13, and P14. No species of the length predicted for a P15-specific subgenomic RNA was detected.

[Marfan syndrome and pregnancy, complicated by dissecting aneurysm]. / Marfan-Syndrom und Schwangerschaft, kompliziert durch das Aneurysma dissecans.

Pregnant women with a Marfan syndrome carry a risk of developing an aortic arc dissection. As the developing aneurism may be diagnosed by echocardiography, this method should be applied repeatedly during pregnancy. We report the case of a Marfan patient, who developed an acute aortic dissection during the third trimester of her pregnancy and discuss the guidance and surveillance of Marfan patients who are or wish to become pregnant.

Ventricular arrhythmias in hypertensive heart disease with and without heart failure.

Forty-two patients with hypertensive heart disease but without coronary macroangiopathy were examined for ventricular arrhythmias by means of 24-h, long-term electrocardiograms (ECG). They were divided into two groups according to specific criteria. Group 1 was composed of 30 patients with left ventricular hypertrophy but normal ventricular volumes, as determined by ventriculography. Group 2 comprised 12 patients with left ventricular hypertrophy and dilated left ventricles. By means of two 24-h, long-term ECGs, the mean absolute number of ventricular extrasystoles was ascertained and severity was determined according to the classification of Ryan et al. On average, patients in group 2 showed 7.830 +/- 6.579 extrasystoles, a significantly higher (p less than 0.001) number than in patients in group 1 who had 1.132 +/- 2.639 extrasystoles/24 h. Moreover, 67% of patients in group 2 had Ryan's class 4a ventricular arrhythmias (couplets) or 4b disorders (ventricular tachycardia). However, corresponding rhythm disorders could be found in only 7% of the patients in group 1. A comparison of hemodynamic parameters and ventricular arrhythmias showed that a decreasing left ventricular ejection fraction (EF, expressed in %), a decreasing mass/volume ratio (LVMM/EDV), and an increasing systolic wall stress of the left ventricle (Tsyst) are accompanied by a nearly linear increase in ventricular extrasystoles and in the severity of the ventricular arrhythmias. During long-term ECGs, nine of 10 patients with systolic wall stress of greater than or equal to 300 dyn x 10(3)/m2 showed Ryan's class 4a or 4b ventricular arrhythmias or ventricular tachycardia during programmed ventricular stimulation. However, 12 patients with normal systolic wall stress (less than or equal to 200 dyn x 10(3)/m2) showed no or only Ryan's class 1 ventricular arrhythmias. Our investigations have shown that cardiac ventricular rhythm disorders frequently occur during decompensated hypertensive heart disease, but to a lesser extent in left ventricular hypertrophy without dilation. Further investigations are needed to demonstrate whether regression of left ventricular hypertrophy is accompanied by a reduction in the incidence of ventricular arrhythmias.