Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle

The mechanisms that determine how folding attempts are interrupted to target folding-incompetent proteins for endoplasmic reticulum-associated degradation (ERAD) are poorly defined. Here the alpha-mannosidase I-like protein EDEM was shown to extract misfolded glycoproteins, but not glycoproteins undergoing productive folding, from the calnexin cycle. EDEM overexpression resulted in faster release of folding-incompetent proteins from the calnexin cycle and earlier onset of degradation, whereas EDEM down-regulation prolonged folding attempts and delayed ERAD. Up-regulation of EDEM during ER stress may promote cell recovery by clearing the calnexin cycle and by accelerating ERAD of terminally misfolded polypeptides.     

Effects of the aglycone of ascaulitoxin on amino acid metabolism in Lemna paucicostata

Ascaulitoxin and its aglycone (2,4,7-triamino-5-hydroxyoctanoic acid, CAS 212268-55-8) are potent phytotoxins produced by Ascochyta caulina, a plant pathogen being developed for biocontrol of weeds. The mode of action of this non-protein amino acid was studied on Lemna paucicostata. Ascaulitoxin is a potent growth inhibitor, with an I₅₀ for growth of less than 1 μM, almost completely inhibiting growth at about 3 μM. Its action is slow, starting with growth inhibition, followed by darker green fronds, and then chlorosis and death. Most amino acids, including non-toxic non-protein amino acids, reversed the effect of the toxin when supplemented in the same medium. Supplemental sucrose slightly increased the activity. d-Amino acids were equally good inhibitors of ascaulitoxin activity, indicating the amino acid effects may not be due to inhibition of amino acid synthesis. Oxaloacetate, the immediate precursor of aspartate, also reversed the activity. LC-MS did not detect interaction of the compound with lysine, an amino acid that strongly reversed the effect of the phytotoxin. Metabolite profiling revealed that the toxin caused distinct changes in amino acids. Reduction in alanine, paralleled by enhanced levels of the branched chain amino acids valine, leucine and isoleucine and nearly unchanged levels of pyruvate, might indicate that the conversion of pyruvate to alanine is affected by ascaulitoxin aglycone. In addition, reduced levels of glutamate/glutamine and aspartate/asparagine might suggest that synthesis and interconversion reactions of these amino group donors are affected. However, neither alanine aminotransferase nor alanine: glyoxylate aminotransferase were inhibited by the toxin in vitro. Our observations might be explained by three hypotheses: (1) the toxin inhibits one or more aminotransferases not examined, (2) ascaulitoxin aglycone affects amino acid transporters, (3) ascaulitoxin aglycone is a protoxin that is converted in vivo to an aminotransferase inhibitor.     

Assessment of geographic variation by RAPD markers among Italian open-pollinated progenies of Populus alba L.

Random amplified polymorphic DNA (RAPD) analysis wasapplied to evaluate variation among provenances of white poplar(Populus alba L.) collected indifferent geographic areas in Italy. Fifteen decamer oligonucleotidesamplified a total of 71 distinct fragments of which 57(80% of the total observed) were polymorphic among53 plants of white poplar. Among provenances, the highest degree ofpolymorphism was detected in the population from Sinni river(62%), while the lowest level was recorded in thepopulation from Arno river (42%). Cluster(UPGMA) analysis was performed on the RAPD data and itdifferentiated the analysed plants in three distinct groupscorresponding to the Apennine, Southern and Northern Italian areas.The RAPD clusters were congruent with groups based on phenologicaltraits.     

Antioxidants, free radicals, storage proteins, puroindolines, and proteolytic activities in bread wheat (Triticum aestivum) seeds during accelerated aging

Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.     

Intergeneric relationships in the Australian Vitaceae: new evidence from cpDNA analysis

Taxa related to important agricultural species are likely to contain a considerable amount of potentially valuable genetic diversity. Nevertheless, before breeding programs or gene discovery projects can be initiated it is important to understand the phylogenetic relationships between the species involved. A component of a major gene discovery project in grapes at the Centre for Plant Conservation Genetics (Southern Cross University, Australia) is directed at the discovery of novel genes in native Vitaceae. As a result a study was conducted in order to assess the phylogenetic relationships between V. vinifera and the native members of the three major Australian genera: Cayratia, Cissus and Tetrastigma. CpDNA sequence analysis (from the trnL intron) adequately resolved intergeneric relationship between the majority of the species studied and provided some useful new information on the phylogenetic relationships within the Vitaceae. This preliminary project identified two species, C. hypoglauca and C. sterculiifolia, as being closely related to V. vinifera and worthy of further in-depth investigation.     

Determination of choline in milk, milk powder, and soy lecithin hydrolysates by flow injection analysis and amperometric detection with a choline oxidase based biosensor

A fast-response and interference-free amperometric biosensor based on choline oxidase immobilized onto an electropolymerized polypyrrole film for flow injection determination of choline in milk, milk powder, and soy lecithin hydrolysates is described. The sensor displayed an Imax value of 1.9 +/- 0.2 microA and an apparent Michaelis-Menten constant, k'M, equal to 1.75 +/- 0.07 mM. Detection limits of 0.12 microM could be obtained. Because even a slight deterioration of the anti-interference membrane can adversely affect measurement accuracy, a real time monitoring of the biosensor selectivity has been achieved by a dual Pt electrode flow-through cell where the enzyme modified electrode is coupled to an enzyme-free electrode in a parallel configuration. Finally, bracketing technique (alternate injections of sample and standards) allows a two-point calibration to be performed in real-time, correcting for any drift in sensor response.     

On the chemical composition and nutritional value of pleurotus taxa growing on umbelliferous plants (apiaceae)

Unpublished data on the chemical composition and nutritional value of Pleurotus mushrooms, growing on Umbelliferous plants (Apiaceae), are here reported. Cultivated basidiomata of four different Pleurotus taxa were analyzed in order to evaluate the composition in lipids, sugars, nitrogen, water, vitamins, ashes, and energetic values. The results showed that Pleurotus mushrooms are suitable in every type of diet thanks to their low caloric content, gastronomic value, vitamins, and mineral salt contents. The presence of a high content of vitamin B(12) and riboflavin in Pleurotus nebrodensis is noteworthy.     

The impact of clonality on an endangered tree (Elaeocarpus williamsianus) in a fragmented rainforest

The occurrence and distribution of clonality in the endangered rainforest tree Elaeocarpus williamsianus (Elaeocarpaceae) was investigated using SSR and RAPD analyses for 170 apparent individual trees found across seven sites. The results obtained with the two molecular techniques were in complete agreement in showing that single clones are present in most of the E. williamsianus sites with two genets occurring at the largest and most adequately protected site. In addition, seed production, viability and germinability were determined for four of these populations. Fruit were produced in all four populations tested although sterile fruit were very common. Only two E. williamsianus trees representing different genets within the same site produced viable seed. The overall genetic diversity within E. williamsianus is much lower than expected and thus the potential for sexual reproduction has been significantly diminished. It is concluded that habitat fragmentation removed an existing balance between vegetative and sexual reproduction in this species. Such findings have added urgency to the management of this species which could include a reintroduction program incorporating the collation of all clones within selected sites.     

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.     

Evaluation of phenolic compounds in virgin olive oil by direct injection in high-performance liquid chromatography with fluorometric detection

Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenyl alcohols, phenolic acids, secoiridoids such as the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol or (p-hydroxypheny1)ethanol (3,4-DHPEA-EDA or p-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA), lignans such as (+)-1-acetoxypinoresinol and (+)-pinoresinol, and flavonoids. A new method for the analysis of VOO hydrophilic phenols by direct injection in high-performance liquid chromatography (HPLC) with the use of a fluorescence detector (FLD) has been proposed and compared with the traditional liquid-liquid extraction technique followed by the HPLC analysis utilizing a diode array detector (DAD) and a FLD. Results show that the most important classes of phenolic compounds occurring in VOO can be evaluated using HPLC direct injection. The efficiency of the new method, as compared to the liquid-liquid extraction, was higher to quantify phenyl alcohols, lignans, and 3,4-DHPEA-EA and lower for the evaluation of 3,4-DHPEA-EDA and p-HPEA-EDA.