Acyl-glucose-dependent glucosyltransferase catalyzes the final step of anthocyanin formation in Arabidopsis

The biosynthetic pathways that produce anthocyanins, the principal pigments for flower and leaf coloration in plants, have been extensively investigated. As a result, many of the enzymes involved in these pathways have been identified. Here, we make use of an inducible Arabidopsis thaliana system and demonstrate that the final step in the formation of the major anthocyanin molecule occurs via a glucosylation step catalyzed by acyl-glucose-dependent anthocyanin glucosyltransferase (AAGT). The glucosylation occurs at the 4-coumarate moiety of the anthocyanin molecule cyanidin 3-O-[2″-O-(2′″-O-(sinapoyl) xylosyl) 6″-O-(p-coumaroyl) glucoside] 5-O-[6″″-O-(malonyl) glucoside] leading to completion of the main anthocyanin structure, a reaction that has not previously been identified in studies of Arabidopsis anthocyanins. Earlier studies on flower AAGTs showed that they conjugate a glucose directly to the basic skeleton of anthocyanin. The present study provides the first evidence that an AAGT of Arabidopsis can conjugate a glucose to an acyl moiety of an anthocyanin modified with sugars and organic acids. The results from analyses of gene expression and of anthocyanin composition in a knock-out (KO) mutant and from a complementation test indicate that AtBGLU10 might encode this AAGT.     

Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco

Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.     

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin

Background

Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity.

Methods

Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured.

Results

A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25 μM and 5 to 25 μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900 μM for their EC₅₀, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells.

Conclusions

These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections.

General significance

Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.     

A novel COL7A1 gene mutation causing pretibial epidermolysis bullosa: Report of a Chinese family with intra-familial phenotypical diversity

Pretibial epidermolysis bullosa (PEB) is an extremely rare subtype of dominant dystrophic epidermolysis bullosa (DDEB) caused by mutation of the COL7A1 gene. More than 730 mutations have been identified in patients with DDEB, but only five mutations have been found to be related to PEB. In this study, a novel heterozygous nucleotide G > T transition at position 6101 in exon 73 of COL7A1 was detected, which resulted in a glycine to valine substitution (G2034V) in the triple-helical domain of type-VII collagen. This is the first report to show that one mutation caused a broad range of severity of disease in one family with PEB. These data suggest that c.6101G > T may influence the phenotype of PEB. They also contribute to the expanding database on COL7A1 mutations.     

Collaborative grid infrastructure for molecular simulations: The eMinerals minigrid as a prototype integrated compute and data grid

This paper describes a prototype grid infrastructure, called the “eMinerals minigrid”, for molecular simulation scientists. which is based on an integration of shared compute and data resources. We describe the key components, namely the use of Condor pools, Linux/Unix clusters with PBS and IBM's LoadLeveller job handling tools, the use of Globus for security handling, the use of Condor-G tools for wrapping globus job submit commands, Condor's DAGman tool for handling workflow, the Storage Resource Broker for handling data, and the CCLRC dataportal and associated tools for both archiving data with metadata and making data available to other workers.     

Validation of Monte carlo Geant4 multithreading code for a 6 MV photon beam of varian linac on the grid computing

AimTo evaluate the computation time efficiency of the multithreaded code (G4Linac-MT) in the dosimetry application, using the high performance of the HPC-Marwan grid to determine with high accuracy the initial parameters of the 6 MV photon beam of Varian CLINAC 2100C.BackgroundThe difficulty of Monte Carlo methods is the long computation time, this is one of the disadvantages of the Monte Carlo methods.Materials and methodsCalculations are performed by the multithreaded code G4Linac-MT and Geant4.10.04.p02 using the HPC-Marwan computing grid to evaluate the computing speed for each code. The multithreaded version is tested in several CPUs to evaluate the computing speed according to the number of CPUs used. The results were compared to the measurements using different types of comparisons, TPR_20.10, penumbra, mean dose error and gamma index.ResultsThe results obtained for this work indicate a much higher computing time saving for the G4Linac-MT version compared to the Geant4.10.04 version, the computing time decreases with the number of CPUs used, can reach about 12 times if 64CPUs are used. After optimization of the initial electron beam parameters, the results of the dose simulations obtained for this work are in very good agreement with the experimental measurements with a mean dose error of up to 0.41% on the PDDs and 1.79% on the lateral dose.ConclusionsThe gain in computation time leads us to perform Monte Carlo simulations with a large number of events which gives a high accuracy of the dosimetry results obtained in this work.     

Isolation,Purification, and Physical and Chemical Properties of Neuraminidase Inhibitor No. 289

The neuraminidase inhibitor produced by Streptomyces sp. No. 289 has been isolated from a culture filtrate and purified, and the properties of the purified preparation have been investigated. The inhibitor has a molecular weight of about 100,000, being free from neuraminic acid or its analogs and consisting of 88% of sugar and 12% of protein. The sugar constituent is mainly composed of equal amounts of glucose and mannose, and the protein constituent lacks S-containing amino acids. An elementary analysis gives 37.33% C, 6.12% H and 1.29% N. The activity of the inhibitor is stable to heating at 100°C for 10 min and to the actions of various proteolytic enzymes, but is weakened by periodate oxidation. These properties have proved that the inhibitor is completely different from those so far reported.     

Cobalt(III)-induced hexamerization of PEGylated insulin

Insulin conjugates in which the B1Phe residue has been chemically modified often exhibit a reduced tendency to associate into hexamers due to weakened interactions between subunits. The purpose of this study was to prepare a hexamer formulation for such insulin conjugates by using Co(III) as a coordinating metal ion. PEGylated insulin in which monomethoxypoly(ethylene glycol) (mPEG, M_r 5000 or 20,000) had been site-specifically attached to B1Phe was chosen as a model conjugate. Hexamerization of mPEG-insulin upon H₂O₂-mediated oxidation of Co(II) was kinetically and quantitatively analysed by visible spectrometry and size-exclusion HPLC. Co(III) mPEG-insulin hexamers thus obtained were extremely stable, existing mostly as a hexameric form even at nanomolar concentrations. A remarkable increase in hydrodynamic volumes was observed for Co(III) mPEG(20k)-insulin hexamers (1600 kDa), as well as Co(III) mPEG(5k)-insulin hexamers (300 kDa). Our results demonstrate the potential benefits of Co(III) hexamer formulation for weakly associating insulin conjugates in the treatment of diabetes.     

Green tea polysaccharide-conjugates protect human umbilical vein endothelial cells against impairments triggered by high glucose

Hot-water extracts of low-grade green tea were precipitated with ethanol, deproteinized with trichloroacetic acid, neutralized with NaOH and fractionated by DEAE-cellulose DE-52 column chromatography to yield three (3) of unexplored polysaccharide-conjugate fractions termed gTPC1, gTPC2 and gTPC3. Monosaccharide and amino acid composition, contents of total neutral sugars, proteins and moistures, HPGPC distribution and Zeta potentials of gTPC1-3 were investigated. Exposure of human umbilical vein endothelial (HUVE) cells to high glucose (33 mM) for 12 h significantly decreased cell viability relative to normal glucose control (p < 0.001). As compared with cell injury group, gTPC1-3 at all of three dose levels (50, 150 and 300 μg/mL) were found to possess remarkably protective effects on HUVE cells against impairments induced by high glucose in a dose-dependent manner (p < 0.05, p < 0.001). To contribute toward our understanding of the cell-based protection mechanism of gTPC1-3, the latter were subjected to self-oxidation of 1,2,3-phentriol assay, and their scavenging effects were observed as 55.1%, 47.6% and 47.9% at the concentration of 300 μg/mL, respectively. On the basis of the fact that high glucose-induced endothelial dysfunction involves in the overproduction of reactive oxygen species (ROS) and contributes to the vascular complications in patients with diabetes, inhibitory effects of gTPC1-3 on high glucose-mediated HUVE cell loss are, at least in part, correlated with their potential scavenging potency of ROS. Taken together, gTPC1-3 could be developed as non-cytotoxic candidates of therapeutic agent for diabetic vascular complications.