Taraxacum officinale and related species-An ethnopharmacological review and its potential as a commercial medicinal plant.

ETHNOPHARMACOLOGICAL RELEVANCE: Dandelion (Taraxacum spec) is a wild plant that has been used for centuries as a traditional medicine in the relief and treatment of several diseases. This use is due to the presence of sesquiterpenes, saponins, phenolic compounds, flavonoids, and sugars, among others, found in the organs of the plant. AIM OF THE STUDY: The aim of this work is to provide a current review of developments and trends in research on the Taraxacum genus, with a focus on traditional uses and pharmacological properties. This should shed light on the potential of this plant as an attractive commercial herbal medicine. MATERIALS AND METHODS: Documents were collected, analyzed, and classified for information regarding medical, agronomic, genetic, and biological aspects of the Taraxacum species. This process was based on a thorough search of documents indexed by scientific search engines. RESULTS: Two important periods of research on Taraxacum have been identified: the first, between 1930 and 1950; and the second, from 1990 to today. During the former, agricultural and genetics research on this plant were, due to the shortage of natural rubber, the focus. In contrast, the main drive in Taraxacum research is now the recovery of bioactives and/or applications in medicine. Pharmacology is the main area in which these plants have been tested, thanks in part to its widely known traditional uses; however, there is less than enthusiastic interest in further human clinical trials. In other areas, Taraxacum sports an enormous list of compounds of industrial interest; and while it is true that only a small amount of these compounds is immediately available in Taraxacum organs and makes it relatively commercially unattractive, only scarce efforts have been made to improve yields. Compounding this issue, most studies of its growth and cultivation have been focused mainly on controlling it as a weed detrimental to certain industrial crops. To wit, in spite of all the research carried out, less than 1% of all the species identified so far (>2500) have been studied (including Taraxacum officinale, Taraxacum coreanum, Taraxacum mongolicum and Taraxacum platycarpum). This is a indication of the little knowledge that we have about this genus so far. Biotechnology (involving genetics, agriculture, and biology) is the most powerful means by which to take advantage of all the medicinal potential of Taraxacum. Great strides have been made in identifying metabolic pathways for synthesizing terpenes, one of the most important compound families in clinical applications. In order to improve yield and performance of the plant in the field, greenhouse cultivation is another aspect taken into account, deriving an increase in recovery of bioactives from Taraxacum organs. Even while considering that only a few species have been studied, their different biochemical and cultivation profiles indicate huge potential for qualitative improvements in composition through genetic engineering, thus directly impacting pharmacological properties. CONCLUSIONS: Taraxacum is has been traditionally considered a natural remedy, well-inserted into popular knowledge, but with low commercial applicability. Only once the recovery of pure and highly reactive compounds can be pursued at (a qualitatively and quantitatively attractive) economical scale, human clinical trials would be of interest in order to prove their efficacy and safety, positioning Taraxacum as an important commercial source of natural drugs.

Multiple cis-regulatory elements are involved in the complex regulation of the sieve element-specific MtSEO-F1 promoter from Medicago truncatula.

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1.

Tomato Ve disease resistance genes encode cell surface-like receptors.

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLphi or YXXphi endocytosis signals (phi is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

Novel approach in plastid transformation.

Engineering the nuclear genome of plants is perceived to be associated with problems regarding biosafety and the stability of expression of the transgene. Alternative transformation strategies using the genomic outfit of the plastid promise to be more successful in this respect. Over the past few years progress has been made in screening procedures, and plastid transformation technology has allowed function to be assigned to open reading frames, massive expression of insecticidal agents and proteins involved in herbicide resistance, and the accumulation of biopolymers. Recently, the design of a novel femtoinjection technique that allows injection into chloroplasts has provided the opportunity to further manipulate and understand chloroplastic gene expression.

[In vitro investigation of biological and technical prosthetic heart valves using MRI: evaluation of possible deflection and heating of the implants]. / In-vitro-Untersuchung von biologischen und technischen Herzklappenprothesen im MRT: Beurteilung möglicher Anziehung und Erhitzung der Implantate.

PURPOSE: In vitro evaluation of possible deflection and heating of present-day prosthetic heart valves during MR imaging at 1.5 T. METHODS: 17 prosthetic heart valves, 12 technical and 5 biological, were investigated using a 1.5 Tesla Siemens Vision system. Deflection was measured at the edge of a 1.5 Tesla superconducting magnet. Each valve was then submerged in a vial of a 1/1 electrolyte solution and temperature was measured before and after imaging with a turbo-spin-echo sequence (TR 5200 ms, TE 138 ms, Flip angle 180 degrees, acquisition time 10.5 minutes, length of echo train 29). MR imaging was performed with phase encoding parallel and perpendicular to the plane of the valves. RESULTS: None of the investigated prosthetic heart valves were deflected. The maximal observed temperature rise was 0.5 degree C. During MR investigation of the prostheses, artifacts caused by metallic parts were less evident using a spin-echo sequence than a gradient-echo sequence. CONCLUSIONS: Patients with the tested present-day prosthetic heart valves can be safely imaged by MRI.

Immunological analysis of potato leafroll luteovirus (PLRV) P1 expression identifies a 25 kDa RNA-binding protein derived via P1 processing.

Mono- and polyclonal antibodies directed against different domains of the potato leafroll luteovirus (PLRV) P1 (ORF1) protein were applied to the analysis of P1 expression during PLRV replication in planta. Western analyses detected P1 and a protein of approximately 25 kDa (P1-C25) that accumulated to readily detectable amounts in PLRV-infected plants, but was not detected by in vitro cell-free translation of P1. P1-C25 represents the C-terminus of P1 and is a proteolytic cleavage product produced during P1 processing. On the basis of its molecular weight, the N-terminus of P1-C25 is either identical to or located adjacent to the previously identified PLRV genome-linked protein, VPg. P1-C25 is not associated with virus particles, and subcellular localization experiments detected P1-C25, but not P1, in the membrane and cytoplasmic fractions of PLRV-infected cells. In addition, P1-C25 exhibits nucleic acid-binding properties. On the basis of its biosynthesis, localization and biochemical properties, P1-C25 may facilitate the formation of P1/PLRV RNA complexes in which the spatial proximity allows for covalent bond formation between PLRV RNA and VPg.

In vitro effects of anticalcification treatment on the calcium uptake of bioprosthetic materials.

BACKGROUND AND AIM OF THE STUDY: The frequent need for re-replacement of tissue valves due to calcification remains their major disadvantage compared with mechanical implants. A variety of anticalcification treatments has been proposed but none has proved to prevent calcific degeneration. The study aim was to evaluate, in vitro, the time course of calcium uptake of procine and bovine biomaterials and the efficacy of anticalcification treatments. METHODS: Samples of glutaraldehyde-tanned biomaterials with (Carpentier-Edwards, Medtronic Intact, Hancock II, MZGTB) or without anticalcification treatment (Mitroflow) were incubated with culture medium containing physiological calcium levels. Specimens were then analyzed at two, four or eight weeks for their calcium content (mg/g wet weight). RESULTS: All specimens calcified over time, though the time courses for each were significantly different. Mitroflow and Intact valves accumulated high calcium levels rapidly during the first two weeks, with small further increases thereafter. Anticalcification treatment reduced calcium uptake of Carpentier-Edwards material during the first two weeks of incubation, but the material gradually attained comparable calcium levels at eight weeks. Hancock valves and the self-customized MZGTB valve showed the lowest calcium levels over the test period. CONCLUSIONS: This in vitro study documented major differences in calcium uptake of different biomaterials. Some anticalcification treatments render the material temporarily less susceptible to calcium binding but none can prevent calcification. In vitro testing has proved to be a valuable instrument for evaluating anticalcification treatments, but should be combined with evaluation of bioprosthesis surface interaction with circulating blood.

Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves ischemic myocardium from reperfusion injury.

Myocardial injury after ischemia (I) and reperfusion (R) is related to leukocyte activation with subsequent release of cytokines and oxygen-derived free radicals as well as complement activation. In our study, the cardioprotective effects of exogenous C1 esterase inhibitor (C1 INH) were examined in a rat model of myocardial I + R (i.e., 20 min + 24 hr or 48 hr). The C1 INH (10, 50 and 100 U/kg) administered 2 min before reperfusion significantly attenuated myocardial injury after 24 hr of R compared to vehicle treated rats (P < .001). Further, cardiac myeloperoxidase activity (i.e., a marker of PMN [polymorphonuclear leukocyte] accumulation) in the ischemic area was significantly reduced after C1 INH treatment compared to vehicle treated animals (0.81 +/- 0.1, 0.34 +/- 0.13, 0.13 +/- 0.1 vs. 1.44 +/- 0.3 U/100 mg tissue, P < .001). In addition, C1 INH (100 U/kg) significantly attenuated myocardial injury and neutrophil infiltration even after 48 hr of reperfusion compared to vehicle treatment. Immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated activation of classical complement pathway by deposition of C1q on cardiac myocytes and cardiac vessels. In addition, expression of the endothelial adhesion molecules P-selectin and intercellular adhesion molecule 1 (ICAM-1) was observed after reperfusion of the ischemic myocardium. In this regard, C1 INH administration abolished expression of P-selectin and ICAM-1 on the cardiac vasculature after myocardial ischemia and reperfusion. Blocking the classical complement pathway by exogenous C1 INH appears to be an effective means to preserve ischemic myocardium from injury after 24 and 48 hr of reperfusion. The mechanisms of this cardioprotective effect appears to be due to blocking of complement activation and reduced endothelial adhesion molecule expression with subsequent reduced PMN-endothelium interaction, resulting in diminished cardiac necrosis.

Determinants of calcium uptake of bovine pericardium for heart valve replacement: results of in vitro studies.

BACKGROUND AND AIMS OF THE STUDY: Reduction of biomaterial calcification is an important aim in the basic research of biological heart valves. An in vitro model was used to investigate the influence of serum calcium concentration and surface coverage with cells or basal proteins on calcium uptake of bovine pericardium. METHODS: Samples of glutaraldehyde-tanned bovine pericardium, stored in formaldehyde and detoxified with borohydride were incubated for two weeks with cell culture medium containing low (1.0 mmol/l) or physiologic (2.3 mmol Ca/l) calcium concentration. Specimens were either unseeded, completely surface-covered with rat fibrocytes (rf) or fibrin (fi), or incompletely seeded with rabbit cells (re). Quality of surface coverage was assessed by surface scanning electron microscopy and calcium content by atomic absorption spectroscopy. RESULTS: Serum calcium had a significant influence on calcium uptake (low versus physiological (1.58 +/- 2.45 mg/g versus 8.10 +/- 1.73 mg/g wet wt, p < 0.001). This may explain early calcification of bioimplants in children and patients on dialysis. Surface coverage significantly reduces calcium uptake (fi, 1.20 +/- 0.41 mg/g, rf, 4.20 +/- 1.70 mg/g, p < 0.001) but complete coverage is necessary (re, 6.98 +/- 1.64 mg/g, NS). CONCLUSIONS: In vitro testing of calcium uptake has proven to be a valuable tool for evaluation of biomaterial calcification.

Early failure of an autologous pericardium aortic heart valve (ATCV) prosthesis.

BACKGROUND AND AIMS OF THE STUDY: The risk of reoperation due to calcific degeneration is the most important drawback of biological heart valves compared with mechanical prostheses. Concepts to mitigate calcification have been tested experimentally and clinically, but none has proven completely to prevent degeneration. METHODS: Renewed interest has involved the use of autologous pericardium for intraoperative construction of autologous aortic valves (ATCV). RESULTS: Of 10 ATCV implanted between 1994 and 1996, one was removed from an 84-year-old female 27 months after implantation because of severe valvular insufficiency. Eight months earlier, a broken spring of the stent system was detected radiographically, with normal valvular competence at echocardiography. Valve dysfunction was due to shrinkage of one leaflet. Measurement of tissue calcium by atomic absorption spectroscopy showed high levels in the shrunken leaflet compared with two intact leaflets (42.82 versus 0.51 and 2.42 mg Ca2+/g). Histology and immunohistology demonstrated extensive fiber degeneration without inflammation or immune reactions in the destroyed leaflet, and moderate fiber degeneration in intact leaflets. Scanning electron microscopy showed smooth surfaces in the normal leaflet and exposure of collagen in the degenerated leaflet in association with calcium deposition. CONCLUSIONS: In contrast to the outcome in animal studies, intraoperative tanning did not prevent tissue shrinkage in this case. Leaflet malfunction was associated with calcification. At present, the clinical use of valves constructed from autologous pericardium can not be recommended.

In planta transcription of a second subgenomic RNA increases the complexity of the subgroup 2 luteovirus genome.

The genetic information of potato leafroll virus (PLRV), a typical member of the subgroup 2 luteoviruses, is contained in a single-stranded (+) sense RNA of approximately 5.9 kb. A single subgenomic RNA (sgRNA1) of approximately 2.3 kb has been characterized as the mRNA for the 3' clustered viral open reading frames ORF3, ORF3/5 and ORF4. Here we demonstrate by Northern blot analyses of polysomal RNAs from PLRV-infected Solanum tuberosum and Physalis floridana plants that, as with luteoviruses belonging to subgroup 1, in planta synthesis of a second 0.8 kb subgenomic RNA (sgRNA2) increases the complexity of subgroup 2 luteoviral genomes significantly. PLRV-specific hybridization probes as well as primer extension experiments map sgRNA2 to the 3'-end of the PLRV RNA genome (positions 5190-5987). Similarly, for the closely related cucurbit aphid-borne yellows virus (CABYV) a sgRNA2 of similar size and position (positions 4888-5669) was identified. PLRV sgRNA2 may code for two viral proteins of 7.1 (ORF6) and 14 kDa (ORF7) respectively, while the CABYV proteins are 8.7 (ORF6) and 8.3 kDa (ORF7) in size, with PLRV ORF7 displaying nucleic acid binding activity. In vivo experiments by transient expression of chimeric GUS fusions in potato protoplasts demonstrated that sgRNA2 functions as a bicistronic mRNA with high expression of ORF6 and low translational efficiency for synthesis of ORF7.

Pathophysiology of early failure of autologous aortic heart valves (ATCV).

BACKGROUND: Degeneration remains the major drawback of bioprostheses. Among various concepts to mitigate degeneration, the use of autologous pericardium for intraoperative construction of aortic valves (ATCV) was revived recently. Based on in-vivo studies the problem of tissue failure was claimed to be oversome by short immersion in glutaraldehyde. METHODS: Two often ATCV implanted 1994-1996 had to be replaced because of valvular insufficiency due to leaflet shrinkage or tearing. Pathophysiology of failure was evaluated by light microscopy and immune histology, scanning electron microscopy (SEM) and determination of tissue calcium content (AAS). RESULTS: AAS revealed high calcium levels in the shrunken and low levels in the torn leaflets. Histology demonstrated extensive fiber degeneration without inflammation in the destructed and moderate degeneration in the intact leaflets. SEM showed smooth surfaces in the 'normal' and exposure of collagen in the degenerated leaflet associated with calcification. Tears occurred close to the stents. CONCLUSIONS: Failure of ATCV is characterised by either shrinkage and calcification despite a short tanning or by tearing related to the stent design. Clinical use of ATCV cannot be recommended at present.

In situ localization of the putative movement protein (pr17) from potato leafroll luteovirus (PLRV) in infected and transgenic potato plants.

The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.

In vivo expression of a full-length cDNA copy of potato leafroll virus (PLRV) in protoplasts and transgenic plants.

A full-length cDNA copy (PLRVfl) of potato leafroll virus (PLRV) was constructed and examined in vivo for its biological activities by transient expression experiments with plasmid DNA or in vitro transcribed RNA. In addition, PLRVfl cDNA was stably introduced into the genome of potato plants by Agrobacterium-mediated leaf disc transformation. Both transient and stable expression of PLRVfl resulted in the synthesis of genomic and subgenomic PLRV RNAs. Transgenic plants accumulated the 17-kDa movement protein and displayed the typical symptoms of PLRV infection. This is the first example of the constitutive expression of a phloem-limited virus in planta.

The potato leafroll virus 17K movement protein is phosphorylated by a membrane-associated protein kinase from potato with biochemical features of protein kinase C.

The 17 kDa protein (pr17), the phloem-limited movement protein (MP) of potato leafroll luteovirus (PLRV), is associated with membranous structures and localized to plasmodesmata [Tacke et al. (1993) Virology 197, 274-282; Schmitz, J. (1995) Ph.D. Thesis, University of Cologne]. In planta the protein is predominantly present in its phosphorylated form, but it is rapidly dephosphorylated during isolation under native conditions. In an effort to examine the nature of the protein kinase(s) involved in the phosphorylation reaction, pr17 deletion mutants were expressed as fusion proteins in a bacterial expression vector system and tested for their ability to be phosphorylated by potato membrane preparations as well as by commercially available kinases. A fusion protein containing the nucleic acid-binding, basic, C-proximal domain (pr17C1) was identified to be phosphorylated by a Ca2+- and phospholipid-dependent, membrane-associated protein kinase. This protein kinase activity was inhibited by the addition of (19-36) protein kinase C (PKC) inhibitory peptide, known to be a highly specific inhibitor of mammalian PKC. Moreover, also the mammalian PKC from rat was able to phosphorylate pr17 in vitro. The results suggest that phosphorylation of pr17 takes place at membranous structures, possibly at the deltoid plasmodesmata connecting the sieve cell-companion cell complex of the phloem, by the activity of PKC-related, membrane-associated protein kinase activity.

Effects of surface seeding with vital cells on the calcium uptake of biological materials for heart valve replacement.

BACKGROUND AND AIMS OF THE STUDY: Up to 35% of bioprosthetic heart valves need to be replaced during the first decade after implantation because of tissue degeneration or calcification. The aim of the studies was to evaluate the influence of surface seeding with viable cells on the calcium uptake of biomaterials. MATERIALS AND METHODS: Samples of glutaraldehyde tanned bovine pericardium (GBP, n = 52) or porcine valves (GPV, n = 50), or glycerol treated bovine pericardium (GlyBP, n = 35), which had either been rinsed with saline (GBP, n = 30; GPV, n = 26; GlyBP, n = 18) or seeded with rat fibrocytes (GBP, n = 22; GPV, n = 24; GlyBP, n = 17) were incubated with cell culture medium containing physiologic levels of calcium. Similarly treated material was implanted into the abdominal muscles of 88 rats (seeded: GBP, n = 16; GPV, n = 16; GlyBP, n = 14; non-seeded: GBP, n = 6; GPV, n = 8; GlyBP, n = 7). The specimens were analyzed two and four weeks later for their calcium content. RESULTS: Over time untreated GBP and GlyBP calcified in vitro and in vivo while GPV retained low calcium levels in vivo. Surface seeding with rat fibrocytes significantly reduced the calcium accumulation in GBP and GlyBP in vitro and in vivo (p < 0.05). CONCLUSIONS: Seeding with viable cells might be a promising method of reducing calcium accumulation in bovine pericardial implants.

Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence.

The 5' untranslated leader (Omega sequence) of tobacco mosaic virus (TMV) genomic RNA was utilized as a translational enhancer sequence in expression of the 17 kDa putative movement protein (pr17) of potato leaf roll luteovirus (PLRV). In vitro translation of RNAs transcribed from appropriate chimeric constructs, as well as their expression in transgenic potato plants, resulted in the expected wild-type pr17 protein, as well as in larger translational products recognized by pr17-specific antisera. Mutational analyses revealed that the extra proteins were translated by non-canonical initiation at AUU codons present in the wild-type Omega sequence. In the plant system translation initiated predominantly at the AUU codon at positions 63-65 of the Omega sequence. Additional AUU codons in a different reading frame of the Omega sequence also showed the capacity for efficient translation initiation in vitro. These results extend the previously noted activity of the TMV 5' leader sequence in ribosome binding and translation enhancement in that the TMV translation enhancer can mediate non-canonical translation initiation in vitro and in vivo.

Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses.

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii. The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.