Membrane-Associated Heparan Sulfate Proteoglycan Is a Receptor for Adeno-Associated Virus Type 2 Virions

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.     

The proteome and secretome of human arterial smooth muscle cells

Smooth muscle cells (SMCs) play a crucial role in cardiovascular disorders. A differential proteomic approach should help to elucidate SMC dysfunctions involved in these diseases. With this goal in mind, we plotted the first 2-dimensional (2-D) maps of the proteome and secretome of human arterial smooth muscle cell (ASMC). Intracellular and secreted proteins were extracted from a primary culture of SMCs obtained from patients undergoing coronary artery bypass surgery (n = 11) and separated by 2-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two protein patterns specific to the ASMC proteome and secretome, respectively. A total of 121 and 40 distinct intracellular and secreted polypeptide spots, corresponding to 83 and 18 different proteins, respectively, were identified by matrix-assisted laser desorption/ionization mass spectrometry. The 2-D reference maps and database resulting from this study confirm that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.     

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3–5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.     

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.     

Malting and brewing qualities of some Nigerian rice (Oryza sativa L.) varieties and some thoughts on the assessment of malts from tropical cereals

The maiting qualities of six varieties of Nigerian rice were studied and compared with malts from sorghum and barley. A minimum of seven days was necessary for maximum yield of extract from malted rice. The beer brewed from the six rice malts were similar to each other and to those from sorghum and barley in analytical properties. The appropriateness of using methods designed for assessing barley malt should be re-examined for studying malts from tropical cereals. Meanwhile, it should be sufficient to determine the extract and the nitrogen content of the wort using the European Brewing methods to assess a tropical cereal for mall.

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Résumé Les qualités de maltage de six variétés de riz nigérians ont été étudiées et comparées avec des malts de sorghum et d'orge. Un minimum de 7 jours de maltage est necessaire pour le rendement maximum de l'extrait de riz malté. Les bières brassées à partir de ces six malts de riz étalent samblables entre elles et à celles du sorghum et de l'orge quant à leurs propriétés analytiques. Le caractère approprié de l'emploi de méthodes concues pour évaluer le malt d'orge devrait être réexaminé quand il s'agit d'étudler des malts de ceréales tropicales. Entretemps, pour évaluer le potentiel en malt d'une céréale tropicale, ll devrait être suffisant de déterminer l'extrait EBC et le contenu en azote du moüt, tous deux par la méthode de la Convention Européenne Brassicole.

     

Diversity and dynamics of the DNA and cDNA-derived bacterial compost communities throughout the <Emphasis Type="Italic">Agaricus bisporus</Emphasis> mushroom cropping process

The cultivation of Agaricus bisporus involves the conversion of agricultural materials via fermentation into utilisable simple sugars as a nutrient source for the fungal crop during mushroom cropping. However, little is currently known about the role of the bacterial community contributing to the fermentation process. In this investigation we characterised the composition and dynamics of the DNA and cDNA-derived bacterial populations throughout a commercial mushroom cropping process using MiSeq sequencing. Both methods indicated substantial changes in the bacterial community structure after the first flush of the mushroom crop. However, differences were observed between the composition of the bacterial community determined by each of the two methods. The cDNA-derived community indicated that thermotolerant genera with known sulphur-reducing characteristics were highly active up to the first flush. Activity of the phyla Actinobacteria and Firmicutes was observed to increase as fermentation progressed, indicating that the members of these phyla played prominent roles in the conversion of wheat straw into utilisable sugars. The cDNA-derived community comprised genera with roles in the nitrification process that became highly active at post flush 1. Subsequent chemical analysis of extractable nitrate indicated that substantial nitrification occurred up until the termination of the cropping process. This study has demonstrated that a highly dynamic bacterial community is present throughout the mushroom cropping process.     

Peptide backbone chemistry and membrane channel function: effects of a single amide-to-ester replacement on gramicidin channel structure and function

To examine the structural and functional importance of backbone amide groups in ion channels for subunit folding, hydrogen bonding, ion solvation, and ion permeation, we replaced the peptide bond between Val(1) and Gly(2) in gramicidin A by an ester bond. The substitution is at the junction between the two channel subunits, where it removes an intramolecular hydrogen bond between the NH of Gly(2) and the C==O of Val(7) and perturbs an intermolecular hydrogen bond between the C==O of Val(1) in one subunit and the NH of Ala(5) in the other subunit. The substitution thus perturbs not only subunit folding but also dimer assembly, in addition to any effects on ion permeation. This backbone modification has large effects on channel function: It alters channel stability, as monitored by the channel forming ability and channel lifetime, and ion permeability, as monitored by changes in single-channel conductance and cation permeability ratios. In fact, the homodimeric channels, with two ester-containing subunits, have lifetimes so short that it becomes impossible to characterize them in any detail. The peptide --> ester substitution, however, does not affect the basic subunit fold because heterodimeric channels can form between a subunit with an ester bond and a native subunit. These heterodimeric channels, with only a single ester bond, are more easily characterized; the lone ester reduces the single-channel conductance about 4-fold and the lifetime about 200-fold as compared to the native homodimeric channels. The altered channel function results from a perturbation/disruption of the hydrogen bond network that stabilizes the backbone, as well as the membrane-spanning dimer, and that forms the lining of the ion-conducting pore. Molecular dynamics simulations show the expected destabilization of the modified heterodimeric or homodimeric channels, but the changes in backbone structure and dynamics are remarkably small. The ester bond is somewhat unstable, which precluded further structural characterization. The lability also led to a hydrolysis product that terminates with an alcohol and lacks formyl-Val. Symmetric channels formed by the hydrolyzed product again have short lifetimes, but the channels are distinctly different from those formed by the ester gramicidin A. Furthermore, well-behaved asymmetric channels form between the hydrolysis product and reference subunits that have either an L- or a D-residue at the formyl-NH-terminus.