Higher plant glycosyltransferases

Uridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana. A phylogenetic analysis of the conserved amino acids encoded by these Arabidopsis genes reveals the presence of 14 distinct groups of UGTs in this organism. Genes encoding UGTs have also been identified in several other higher plant species. Very little is yet known about the regulation of plant UGT genes or the localization of the enzymes they encode at the cellular and subcellular levels. The substrate specificities of these UGTs are now beginning to be established and will provide a foundation for further analysis of this large enzyme superfamily as well as a platform for future biotechnological applications.     

A multigene family of glycosyltransferases in a model plant,Arabidopsis thaliana

Glycosyltransferases transfer sugars from NDP-sugar donors to acceptors. The multigene family of transferases described in this paper typically transfer glucose from UDP-glucose to low-molecular-mass acceptors in the cytosol of plant cells. There are 107 sequences in the genome of Arabidopsis thaliana that contain a consensus, suggesting they belong to this Group 1 multigene family. The family has been analysed phylogenetically, and a functional genomics approach has been applied to explore the relatedness of sequence similarity to catalytic specificity and stereoselectivity. Enzymes belonging to this class of transferases glycosylate a vast array of acceptors, including natural products such as secondary metabolites and hormones, as well as xenobiotics absorbed by the plant, such as herbicides and pesticides. Conjugation to glucose potentially changes the activity of the acceptor molecule and invariably changes its location within the plant cell. Using the genomics approach described, a platform of knowledge has been constructed that will enable an understanding to be gained on the role of these enzymes in cellular homoeostasis, as well as their activity in biotransformations in vitro that require strict regioselectivity of glycosylation.     

Ribosomal DNA, species structure, and biogeography of the cactophilic yeast Clavispora opuntiae

The ribosomal DNA of the cactophilic yeast species Clavispora opuntiae was studied in order to clarify the global distribution of the yeast. Over 500 strains, including isolates from several new localities worldwide, were characterized by rDNA restriction mapping. An unusual restriction pattern previously encountered only in one strain, from Conception Island in the Bahamas, was found in several Brazilian isolates. Sequences of the D1/D2 and D7/D8 divergent domains of the large subunit (LSU) and of the intergenic spacers (IGS) confirmed that these strains represent a genetically distinct variety of Clavispora opuntiae. This divergence had previously been hypothesized on the basis of reduced genetic recombination in inter-varietal crosses and the presence of a polymorphic ApaI restriction site located in the LSU. The exact position of the ApaI site in the D8 divergent domain and the nature of the variation that it reveals were determined. The complete sequences of 12 intergenic spacers clarified the significance of the species-wide variation uncovered by restriction mapping. Most of the polymorphic sites occur in the IGS1 and IGS2 regions, on either side of the 5S gene, and the variation is largely due to differences in the numbers and the sequences of internal repeats. Two other polymorphic sites are located in the external transcribed spacer (ETS) region. The reliability of various sites as indicators of overall spacer sequence divergence differed from one case to another. Variety-specific probes were devised and used to screen 120 strains for the presence of recombinant rDNA spacers. Three strains gave ambiguous results, but these did not constitute evidence that inter-varietal recombination has taken place in nature. The hypothesis that the global movement of Clavispora opuntiae has been influenced by the worldwide biological control of prickly pear with Cactoblastis cactorum, a moth of Argentinian origin, has received additional support from the demonstration that Argentinian strains have rDNAs similar to those found where the moth has been introduced. A dramatic founder effect was identified in a yeast population collected in cacti (Maui, Hawaii) in a site where the moth had been recently introduced.     

Characterization of the trans-activation properties of equine herpesvirus 1 EICP0 protein

The EICP0 protein of equine herpesvirus 1 (EHV-1) is an early, viral regulatory protein that independently trans-activates EHV-1 immediate-early (IE), early, gamma1 late, and gamma2 late promoters. To assess whether this powerful trans-activator functions in conjunction with three other EHV-1 regulatory proteins to activate expression of the various classes of viral promoters, transient cotransfection assays were performed in which effector plasmids expressing the EICP22, EICP27, and IE proteins were used either singly or in combination with an EICP0 effector construct. These analyses revealed that (i) independently, the EICP0 and IE proteins are powerful trans-activators but do not function synergistically, (ii) the IE protein inhibits the ability of the EICP0 protein to trans-activate the IE, gamma1 late, and gamma2 late promoters, (iii) the EICP22 and EICP0 proteins do not function together to significantly trans-activate any EHV-1 promoter, and (iv) the EICP27 and EICP0 proteins function synergistically to trans-activate the early and gamma1 late promoters. A panel of EICP0 truncation and deletion mutant plasmids was generated and used in experiments to define the domains of the 419-amino-acid (aa) EICP0 protein that are important for the trans-activation of each class of EHV-1 promoters. These studies revealed that (i) carboxy-terminal truncation mutants of the EICP0 protein exhibited a progressive loss of trans-activating ability as increasing portions of the carboxy terminus were removed, (ii) the amino terminus of the EICP0 protein containing the RING finger (aa 8 to 46) and the acidic region (aa 71 to 84) was necessary but not sufficient for activation of all classes of EHV-1 promoters, (iii) the RING finger was absolutely essential for activation of EHV-1 promoters, since deletion of the entire RING finger motif (aa 8 to 46) or a portion of it (aa 19 to 30) completely abrogated the ability of these mutants to activate any promoter tested, (iv) the acidic region contributed to the ability of the EICP0 protein to activate the early and gamma1 late promoters, and deletion of the acidic region enhanced the ability of this mutant to activate the IE promoter, (v) the carboxy terminus (aa 325 to 419), which is rich in glutamine residues, was dispensable for the EICP0 trans-activation function, (vi) a motif resembling a nuclear localization signal (aa 289 to 293) was unnecessary for the EICP0 protein to trans-activate promoters of any temporal class, and (vii) the EICP0 protein was phosphorylated during infection, and deletion of the serine-rich region (aa 210 to 217), a potential site for phosphorylation, reduced by more than 70% the ability of the EICP0 protein to activate the gamma2 late class of promoters.     

Shared Segment Analysis and Next-Generation Sequencing Implicates the Retinoic Acid Signaling Pathway in Total Anomalous Pulmonary Venous Return (TAPVR)

Most isolated congenital heart defects are thought to be sporadic and are often ascribed to multifactorial mechanisms with poorly understood genetics. Total Anomalous Pulmonary Venous Return (TAPVR) occurs in 1 in 15,000 live-born infants and occurs either in isolation or as part of a syndrome involving aberrant left-right development. Previously, we reported causative links between TAVPR and the PDGFRA gene. TAPVR has also been linked to the ANKRD1/CARP genes. However, these genes only explain a small fraction of the heritability of the condition. By examination of phased single nucleotide polymorphism genotype data from 5 distantly related TAPVR patients we identified a single 25 cM shared, Identical by Descent genomic segment on the short arm of chromosome 12 shared by 3 of the patients and their obligate-carrier parents. Whole genome sequence (WGS) analysis identified a non-synonymous variant within the shared segment in the retinol binding protein 5 (RBP5) gene. The RBP5 variant is predicted to be deleterious and is overrepresented in the TAPVR population. Gene expression and functional analysis of the zebrafish orthologue, rbp7, supports the notion that RBP5 is a TAPVR susceptibility gene. Additional sequence analysis also uncovered deleterious variants in genes associated with retinoic acid signaling, including NODAL and retinol dehydrogenase 10. These data indicate that genetic variation in the retinoic acid signaling pathway confers, in part, susceptibility to TAPVR.     

In vitro endothelial cell activation and inflammatory responses in end-stage heart failure.

BACKGROUND: vascular endothelial cell activation and dysfunction are observed in patients with severe heart failure and may contribute to systemic manifestations of this syndrome. It remains unknown whether inflammatory activation of these cells occurs in these patients because of increased circulating proinflammatory mediators. Aim: to determine whether the serum from patients with heart failure possesses a net proinflammatory bioactivity to active proinflammatory pathways in cultured endothelial cells. METHODS: serum was obtained from stable patients with end-stage heart failure undergoing elective cardiac transplantation (Tx) and severely decompensated patients with heart failure requiring emergency left ventricular assist device (LVAD) implantation. Net proinflammatory bioactivity of serum was investigated by monitoring IkappaBalpha degradation and E-selectin expression in cultured human pulmonary artery endothelial cells (HPAEC) following incubation with serum samples. Serum cytokine concentrations were measured by ELISA and neutralizing antibodies were used to determine the role of specific factors in the observed bioactivity. RESULT: serum from both patient groups induced HPAEC IkappaBalpha degradation. Low basal HPAEC E-selectin expression significantly increased following treatment with Tx but not LVAD serum. Serum tumor necrosis factor-alpha (TNF-alpha) and IL-10 concentrations were higher in patients with LVAD than those with Tx, and soluble TNF-alpha receptor expression was high in both groups. Neither TNF-alpha nor IL-10 blocking experiments altered either bioassay result. CONCLUSION: activation of a specific profile of pro- and anti-inflammatory mediators is associated with heart failure resulting in HPAEC nuclear factor (NF)-kappaB activation. However, E-selectin expression is further regulated by unidentified factors. TNF-alpha is upregulated but appears to play no part in NFkappaB activation in these patients. These findings could have important therapeutic implications.     

Numerical simulation of cardiovascular dynamics with different types of VAD assistance

A variety of methods by which mechanical circulatory support (MCS) can be provided have been described. However, the haemodynamic benefits of the different methods have not been adequately quantified. The aim of this paper is to compare the haemodynamic effects of six forms of MCS by numerical simulation. Three types of ventricular assist device (VAD) are studied: positive displacement; impeller and a novel reciprocating-valve design. Similarly, three pumping modes are modelled: constant flow; counterpulsation and copulsation. The cardiovascular system is modelled using an approach developed previously, using the concentrated parameter method by considering flow resistance, vessel elasticity and inertial effects of blood in individual conduit segments. The dynamic modelling of displacement and impeller pumps is represented by VAD inlet/outlet flow-rate changes. The dynamics of the reciprocating-valve pump is modelled with a specified displacement profile. Results show that in each simulation, the physiological variables of mean arterial pressure and systemic flow are adequately maintained. Modulation of the impeller pump flow profile produces a small (5 mmHg) oscillatory component to arterial pressure, whereas the displacement and reciprocating-valve pumps generate substantial arterial pressure and flow pulsatility. The impeller pump requires the least power input, the reciprocating valve pump slightly more, and the displacement pump the most. The in parallel configuration of the impeller and displacement pump designs with respect to the left ventricle provides near complete unloading and can cause the aortic valve to remain closed throughout the entire cardiac cycle with the attendant risk of aortic valve leaflet fusion following prolonged support. The in series configuration of the reciprocating-valve pump avoids this shortcoming but activation must be carefully synchronized to the cardiac cycle to allow adequate coronary perfusion. The reciprocating-valve pump is associated with haemodynamic advantages and a favourable power consumption.     

Endothelium-dependent and -independent relaxation in the forelimb and hindlimb vasculatures of swine

Limb differences in endothelial function exist between arm and leg vasculatures of humans. The current investigation tested the hypothesis that forelimb and hindlimb vasorelaxation are similar in the absence of limb differences in blood pressure. Conduit arteries (brachials/femorals) and second order arterioles were harvested from 22 miniature Yucatan swine. In vitro assessment of vasorelaxation was determined by administering increasing doses of bradykinin (BK), acetylcholine (ACh), and sodium nitroprusside (SNP). The role of the nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways was assessed in conduit arteries but not resistance arterioles through L-NAME (300 microM) and INDO (5 microM) incubation, respectively. The relaxation responses to BK and ACh were similar in brachial and femoral arteries. SNP relaxation response was greater in the brachial compared to femoral arteries. There were also no significant differences in the relaxation responses of second order arterioles of the forelimb and hindlimb to BK, ACh, and SNP. Incubation of conduit arterial rings in L-NAME produced a greater reduction in BK and ACh relaxation in the brachial (approximately 25%) compared to femoral (approximately 13%) arterial rings. The current results of this investigation suggest that the forelimb and hindlimb vasculatures of swine have relatively similar vasorelaxation responses to both endothelium-dependent and -independent vasodilators.     

Tallgrass prairie restoration: implications of increased atmospheric nitrogen deposition when site preparation minimizes adventive grasses

Site preparation designed to exhaust the soil seedbank of adventive species can improve the success of tallgrass prairie restoration. Despite these efforts, increased rates of atmospheric nitrogen (N) deposition over the next century could potentially promote the growth of nitrophilic, adventive species in tallgrass restoration projects. We used a field experiment to examine how N addition affected species composition and plant productivity over the first 3 years of a tallgrass prairie restoration that was preceded by the planting of glyphosate‐resistant crops and multiple applications of glyphosate to exhaust the pre‐existing seedbank. We predicted that N addition would increase the percent cover of adventive plant species not included in the original seeding. Contrary to our prediction, only the cover of native species increased with N addition; native non‐leguminous forbs increased substantially, with Conyza canadensis (a weedy native species not part of the restoration seed mix) exploiting the combination of high N and bare ground in the first year, and non‐leguminous forbs (in particular Monarda fistulosa) and native C₃ grasses, all of which were seeded, increasing with N addition by the third year. Native legumes was the only functional group that exhibited lower cover in N addition plots than in control plots. There was no significant response by native C₄ grasses to N addition, and adventive grasses remained mostly absent from the plots. Overall, our results suggest that site pre‐treatment with herbicide may continue to be effective in minimizing adventive grasses in restored tallgrass prairie, despite future increases in atmospheric N deposition.     

N-glucosylation of cytokinins by glycosyltransferases of Arabidopsis thaliana

Cytokinins are plant hormones that can be glucosylated to form O-glucosides and N-glucosides. The glycoconjugates are inactive and are thought to play a role in homeostasis of the hormones. Although O-glucosyltransferases have been identified that recognize cytokinins, the enzymes involved in N-glucosylation have not been identified even though the process has been recognized for many years. This study utilizes a screening strategy in which 105 recombinant glycosyltransferases (UGTs) of Arabidopsis have been analyzed for catalytic activity toward the classical cytokinins: trans-zeatin, dihydrozeatin, N(6)-benzyladenine, N(6)-isopentenyladenine, and kinetin. Five UGTs were identified in the screen. UGT76C1 and UGT76C2 recognized all cytokinins and glucosylated the hormones at the N(7) and N(9) positions. UGT85A1, UGT73C5, and UGT73C1 recognized trans-zeatin and dihydrozeatin, which have an available hydroxyl group for glucosylation and formed the O-glucosides. The biochemical characteristics of the N-glucosyltransferases were analyzed, and highly effective inhibitors of their activities were identified. Constitutive overexpression of UGT76C1 in transgenic Arabidopsis confirmed that the recombinant enzyme functioned in vivo to glucosylate cytokinin applied to the plant. The role of the N-glucosyltransferases in cytokinin metabolism is discussed.