MATE2 mediates vacuolar sequestration of flavonoid glycosides and glycoside malonates in Medicago truncatula.

The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3'-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage.

Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes.

PAL (L-phenylalanine ammonia-lyase), the first enzyme of phenylpropanoid biosynthesis, is often encoded by multigene families in plants. A PCR-based approach was used to isolate cDNA clones corresponding to the four PAL genes of tobacco (Nicotiana tabacum). By careful comparison of cDNA and genomic clones, a new PAL gene (PAL4) was defined. PCR amplification of PAL sequences from cDNA led to the generation of chimaeric clones between PAL1 and PAL4, and incorrect annotation of PAL4 ESTs (expressed sequence tags) as PAL1 in the EST database has given rise to a randomly shuffled tentative consensus sequence. The PAL2 previously described in the literature was shown, by domain swapping experiments with PAL1, to possess a single nucleotide substitution leading to an inactive enzyme. The altered amino acid resulting from this substitution maps to the base of the active site pocket in the three-dimensional structure of PAL. The inactive PAL2 allele could not be recovered from 13 different tobacco cultivars examined. PALs 1-4 were co-expressed in multiple plant organs, and were also co-induced following exposure of cell cultures to yeast elicitor or methyl jasmonate. All four tobacco PAL proteins expressed in Escherichia coli displayed normal Michaelis-Menten kinetics, with Km values between 36 and 60 muM. Co-expression of different PAL proteins in E. coli resulted in formation of heterotetramers, which possessed kinetic properties within the same range as those of the individual homotetramers. The potential physiological function of heterotetrameric PAL forms is discussed.

Neuroendocrine mechanisms of left ventricular dysfunction stimulated by anger stress in rats with atherosclerosis--a putative role of natriuretic peptide.

OBJECTIVE: To investigate the role of natriuretic peptide in the process of left ventricular dysfunction caused by emotional stress. METHODS: Adult male SD rats (n=30) and Wistar rats (n=60) were selected in this study. Atherosclerosis models were induced with high-fat diet and excess VD3 injection (eight consecutive weeks), and anger stress models were prepared by resident-intruder stress experiment (two consecutive weeks). Furthermore, left ventricular functions were examined by high-resolution echocardiograph, after which left ventricular myocardium and coronary arteries were prepared for pathological section and observed with electron microscope. At the same time, the hypothalamus, medulla oblongata and left ventricular myocardium were also prepared for pathological sections to detect the localization and expression of ANP, BNP and NPR-A with immunofluorescence and western blot. RESULTS: We found that left ventricular functions of atherosclerosis or emotional stress modeled rats were both inferior to the healthy ones and superior to the combined (atherosclerosis and emotional stress) modeled ones (P<0.05). We also found that atherosclerosis and emotional stress could both cause morphological changes of left ventricular cells and capillary which contribute to apoptosis and hyperblastosis. Further more, there was NPR-A distributed in hypothalamus, medulla oblongata, as well as left ventricular tissues with the same express trend between groups, with atherosclerosis modeled rats the highest and the healthy rats the lowest. CONCLUSIONS: The results of our study suggest that anger stress could cause an excess consumption of ANP, BNP and NPR-A in nervous and cardiovascular system which inhibit the compensatory self-repair function of atherosclerosis rats, leading to a promotion of fibrosis and lipid peroxidation, offering insight into the neuroendocrine mechanisms of left heart function obstacle.

Cardiomyocyte progenitors in a canine pulmonary vein model of persistent atrial fibrillation.

BACKGROUND: This study aimed to discover the pathogenesis of focal atrial fibrillation (AF) originating from pulmonary veins by observing the histological structure and special cells in the canine pulmonary vein model of persistent atrial fibrillation. METHODS: The pulmonary veins and the sinus node were obtained from 10 mongrel dogs (5 AF and 5 control group). Light microscopy, transmission electron microscopy, and immunohistochemistry were applied to transverse sections of each pulmonary vein and sinoatrial node. Morphological and distribution analyses were performed manually and automatically. RESULTS: Cardiomyocyte progenitor (CMPs) and interstitial cells of Cajal (ICCs) showing typical features of either very immature or developing cells were found in the pulmonary vein sections of all animals subjected to experimental AF but not in the control group. The cells were mainly identified in sections with a thick muscular sleeve. A positive immunostaining of CMPs was also demonstrated; the staining characteristic was similar to that of P cells in the sinoatrial node, suggesting that these cells may function in a pacemaker capacity. CONCLUSIONS: We demonstrated that pulmonary veins can host cardiac stem cell niches. Continuous rapid pacing can induce the differentiation of CMPs and ICCs, and CMPs may underlie the pacemaker activity of isolated pulmonary veins.

The effect of early and intensive statin therapy on ventricular premature beat or nonsustained ventricular tachycardia in patients with acute coronary syndrome.

BACKGROUND: To evaluate the prognostic value of early and intensive lipid-lowering treatment on ventricular premature beat or nonsustained ventricular tachycardia (NSVT) after acute coronary syndrome (ACS) (ST-elevation myocardial infarction [STEMI], non-STEMI, and unstable angina pectoris). HYPOTHESIS: Provided that early and intensive lipid-lowering treatment can reduce ventricular premature beat or non-sustained ventricular tachycardia after ACS. METHODS: A total of 586 patients with ACS were randomly divided into 2 groups: group A (with conventional statin therapy, to receive 10 mg/day atorvastatin, n = 289) and group B (early and intensive statin therapy, 60 mg immediately and 40 mg/day atorvastatin, n = 297). The frequency of ventricular premature beat and NSVT was recorded with Holter monitoring after hospitalization (24 hours and 72 hours). RESULTS: Seventy-seven (11.8%) patients had NSVT. When compared to patients with no documented NSVT, patients with NSVT were older and more often had myocardial infarction, diabetes mellitus, atrial fibrillation, and an ejection fraction < 40% in their history. Ventricular premature beats decreased significantly in the early and aggressive treatment group (24 hours, P < 0.01; 72 hours, P < 0.001). A significant reduction in NSVT was seen in the early and aggressive (24 hours, P < 0.01; 72 hours, P < 0.001) group. No side effects were observed in either group. CONCLUSIONS: Early and intensive lipid-lowering treatment can obviously decrease ventricular premature beats and NSVT.

The effect of early and intensive statin therapy on ventricular premature beat or non-sustained ventricular tachycardia in patients with acute coronary syndrome.

BACKGROUND: Our study's aim was to evaluate the prognostic value of early and intensive lipid-lowering treatment on ventricular premature beat or non-sustained ventricular tachycardia (NSVT) after acute coronary syndrome (STEMI, non-STEMI, and unstable angina pectoris). METHODS: Some 586 patients with acute coronary syndrome were randomly divided into two groups: Group A (with conventional statin therapy, to receive 10 mg/day atorvastatin, n = 289) and Group B (given early and intensive statin therapy, 60 mg immediately and 40 mg/day atorvastatin, n = 297). The frequency of ventricular premature beat and NSVT was recorded via Holter monitoring after hospitalization (24 h and 72 h). RESULTS: Seventy seven (11.8%) patients had NSVT. When compared to patients with no documented NSVT, patients with NSVT were older and more frequently had myocardial infarction in their history, diabetes mellitus, atrial fibrillation and an ejection fraction < 40%. Ventricular premature beats decreased significantly in the early and aggressive treatment group (24 h, p < 0.01; 72 h, p < 0.001). A significant reduction in NSVT was seen in the early and aggressive treatment group (24 h, p < 0.01; 72 h, p < 0.001). There were no side effects observed in either group. CONCLUSIONS: Early and intensive lipid-lowering treatment can clearly decrease ventricular premature beats and NSVT.

Regioselective synthesis of plant (iso)flavone glycosides in Escherichia coli.

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4'-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.

Study on the spatial distribution pattern of Cx40 gap junctions in the atria of patients with coronary heart disease.

BACKGROUND: The aim of this study was to evaluate the association between atrial fibrillation and atrial dilation and the spatial distribution pattern of connexin 40 in the atria of patients with coronary heart disease. METHODS: Twenty-six patients with coronary heart disease undergoing cardiac surgery for coronary artery bypass graft were investigated and were divided into three groups according to the left atrial size and rhythm, atrial fibrillation and left atrial dilatation (AF+AD), sinus rhythm and left atrial dilation (SR+AD) and sinus rhythm as control group SR. The spatial distribution patterns of Cx40 were evaluated using confocal laser scanning microscopy assay. RESULTS: No significant differences were observed in the size and density of Cx40 gap junction in the right atrium among all three groups (p > 0.05). Compared with the control group, the size of Cx40 disk area in termination links and in lateral abutment in left atrium was markedly larger in AF+AD group and SR+AD group than those of the controls (p < 0.01). A comparison of size and density of Cx40 gap junction in the left atrium in the AF+AD group and SR+AD group did not show significant differences. CONCLUSIONS: The present study has shown altered gap junction distribution in coronary heart disease resulting from atrial dilation and atrial fibrillation. A decrease in the size and density of Cx40 gap junction was observed in patients with atrial dilation, which could be an important factor in the initiation and maintenance of atrial fibrillation.

Mutational analysis of the Medicago glycosyltransferase UGT71G1 reveals residues that control regioselectivity for (iso)flavonoid glycosylation.

The plant glycosyltransferase UGT71G1 from the model legume barrel medic (Medicago truncatula) glycosylates flavonoids, isoflavonoids, and triterpenes. It can transfer glucose to each of the five hydroxyl groups of the flavonol quercetin, with the 3'-O-glucoside as the major product, and to the A-ring 7-hydroxyl of the isoflavone genistein. The sugar donor and acceptor binding pockets are located in the N and C termini, respectively, of the recently determined crystal structure of UGT71G1. The residues forming the binding pockets of UGT71G1 were systematically altered by site-directed mutagenesis. Mutation of Phe148 to Val, or Tyr202 to Ala, drastically changed the regioselectivity for quercetin glycosylation from predominantly the 3'-O-position of the B-ring to the 3-O-position of the C ring. The Y202A mutant exhibited comparable catalytic efficiency with quercetin to the wild-type enzyme, whereas efficiency was reduced 3-4-fold in the F148V mutant. The Y202A mutant gained the ability to glycosylate the 5-hydroxyl of genistein. Additional mutations affected the relative specificities for the sugar donors UDP-galactose and UDP-glucuronic acid, although UDP-glucose was always preferred. The results are discussed in relation to the design of novel biocatalysts for production of therapeutic flavonoids.

KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen.

The KAP-2 protein that binds to the H-box (CCTACC) element in the bean CHS15 chalcone synthase promoter was purified, and internal peptide sequence used to design primers leading to the cloning of KAP-2 from bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula). KAP-2 shares sequence similarity to the large subunit of mammalian Ku autoantigen, a protein proposed to be involved in control of DNA recombination and transcription. KAP-2 sequences were present in genomic DNA from a range of legumes, and a related protein is found in Arabidopsis thaliana. Recombinant KAP-2 expressed in insect cells showed the same binding specificity for the CHS15 H-box as the protein purified from bean cell extracts. In vitro transcription assays confirmed that KAP-2 stimulates transcription from a promoter harboring the H-box cis element.