Laboratory studies on secondary organic aerosol formation from crude oil vapors.

Airborne measurements of aerosol composition and gas phase compounds over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico in June 2010 indicated the presence of high concentrations of secondary organic aerosol (SOA) formed from organic compounds of intermediate volatility. In this work, we investigated SOA formation from South Louisiana crude oil vapors reacting with OH in a Potential Aerosol Mass flow reactor. We use the dependence of evaporation time on the saturation concentration (C*) of the SOA precursors to separate the contribution of species of different C* to total SOA formation. This study shows consistent results with those at the DWH oil spill: (1) organic compounds of intermediate volatility with C* = 10(5)-10(6) µg m(-3) contribute the large majority of SOA mass formed, and have much larger SOA yields (0.37 for C* = 10(5) and 0.21 for C* = 10(6) µg m(-3)) than more volatile compounds with C*≥10(7) µg m(-3), (2) the mass spectral signature of SOA formed from oxidation of the less volatile compounds in the reactor shows good agreement with that of SOA formed at DWH oil spill. These results also support the use of flow reactors simulating atmospheric SOA formation and aging.

Reflection across plant cell boundaries in confocal laser scanning microscopy.

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.

Preclinical safety testing of DISC-hGMCSF to support phase I clinical trials in cancer patients.

BACKGROUND: DISC-hGMCSF is a gH-deleted HSV-2 based vector expressing human GM-CSF that is being developed for cancer immunotherapy. To support first clinical use, a range of preclinical safety studies were performed using DISC-hGMCSF in addition to DISC-murine-GMCSF and the backbone vector, TA-HSV. METHODS: The toxicity of the DISC vectors was assessed by repeated dose, neurovirulence and neuroinvasiveness studies in mice, and by safety studies in rabbits, guinea pigs and athymic nude mice. Studies were also conducted to determine whether the vector could establish latency in local ganglia in mice following intradermal injection, and whether it could reactivate from the latent state. The vector biodistribution following intravenous administration was also investigated in mice, using PCR to detect vector DNA. RESULTS: The DISC vectors were essentially non-toxic in all the systems studied. No adverse reactions were seen in mice receiving four intravenous doses of DISC-mGMCSF and the results from studies of neurovirulence, neuroinvasiveness, local tolerance in rabbit, general safety in mice and guinea pigs and safety in athymic nude mice were consistent with DISC being unable to replicate and cause disease. The vector could establish latency in local ganglia in mice, but at low efficiency, and could not reactivate infectious virions. Following intravenous administration, vector DNA was widely distributed up to Day 28, but by Day 56 had disappeared from gonads and brain and was only found in blood and liver. CONCLUSION: The panel of safety studies provided evidence that DISC-hGMCSF will be unable to replicate and cause disease, and has low toxicity in man. These data were presented to the Medicines Control Agency and the Gene Therapy Advisory Committee as part of the regulatory submissions for a clinical trial in melanoma patients. These submissions have been approved, and DISC-hGMCSF has now entered a phase I clinical trial in the UK by direct intratumoural injection.

Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules.

Biological nitrogen fixation involves the reduction of atmospheric N2 to ammonia by the bacterial enzyme nitrogenase. In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome. The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant. This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes. This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport.

Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules.

Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts. A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean. GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane. [14C]methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4^{+ channel described elsewhere. GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host.}

Differential expression of the multigene family encoding the soybean mitochondrial alternative oxidase.

The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced for cDNA analysis, were found to be 38.1 and 36.4 Kd, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 KD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.

How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.