Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.     

Interaction of oddball probability and primary task type on P300 in the dual-task paradigm

Using a dual-task paradigm with an oddball secondary task, P300 amplitude and latency were studied as a function of factorially manipulated oddball probability (low = .22, high = .44) and primary task type. In addition to a Baselinecondition (oddball task only), three primary tasks were used: (1) Pure Sensory;watching a movie; (2) Pure Motor (manipulating a flashlight); and (3) Sensory/Motor(using the flashlight to trace the outlines of characters in a movie). The findings included the usual significant effects of probability on amplitude. There was also a significant effect of task type on amplitude, and a significant interaction of oddball probability with task type. In the low but not high probability condition, a pure Sensory task depressed P300 amplitude. In both probability conditions, the Sensory/motortask depressed P300 amplitude. Only task type had a significant effect on P300 latency. The results confirm the ability of other labs (using Sensory/motor primary tasks) to demonstrate P300 depression at high oddball probability, in view of the difficulty in our lab of achieving P300 depression with pure sensory tasks and high oddball probabilities. The results are discussed in terms of partial overlap of processing resource pools. A preliminary report of these data was presented at the 1990 meetings of the Society for Psychophysiological Research.     

Improved coronary vascular function evoked by high-intensity treadmill training is maintained in arteries exposed to ischemia and reperfusion.

We hypothesized that myocardial contractile function and coronary arterial function are greater after ischemia and reperfusion in high-intensity treadmill-trained vs. sedentary rats. Rats performed 10 x 4-min bouts of treadmill running consisting of 2 min at 13 m/min + 2 min at 45-60 m/min (Etr) or were sedentary (Sed) for 12 wk. Animals then were instrumented to measure left ventricular (LV) contractility in response to three 15-min coronary occlusion (O) and 5-min reperfusion (R) cycles (Isc) or a sham operation (Sham). After the Isc and Sham protocols, hearts were excised and coronary arterial ( approximately 105 microm ID) function was evaluated by using isometric techniques. LV developed pressure, the first derivative of LV pressure at a developed pressure of 40 mmHg, and systolic blood pressure were not different between Etr (n = 14) and Sed (n = 7) rats before or after the Sham protocol. Furthermore, hemodynamic variables were similar in Etr (n = 14) and Sed (n = 13) animals before the Isc protocol and were depressed to the same degree by the three O-R cycles. Therefore, Etr did not alter myocardial contractile function in rats that were (i.e., Isc) or were not (i.e., Sham) exposed to ischemia and reperfusion. Acetylcholine-evoked relaxation (10-8 to 3 x 10-5 M) was greater (P < 0.05) in coronary arteries from Sham-Etr vs. Sham-Sed animals (5 of 8 doses tested) and Isc-Etr vs. Isc-Sed rats (3 of 8 doses tested). Maximal relaxation produced by sodium nitroprusside (10-4 M) was similar among groups. Vasocontractile responses produced by KCl (10-100 mM) and endothelin-1 (10-11-10-4 M) were greater (P < 0.05) in the presence vs. the absence of nitric oxide synthase inhibition (10-6 M NG-monomethyl-l-arginine) in vessels from Sham-Etr but not Sham-Sed rats and from Isc-Etr but not Isc-Sed rats. These findings suggest that Etr-evoked improvements in coronary function are maintained in small arteries even when exposed to ischemia and reperfusion.     

Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.     

Diversity of Sulfate-Reducing Bacteria in Oxic and Anoxic Regions of a Microbial Mat Characterized by Comparative Analysis of Dissimilatory Sulfite Reductase Genes

Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the δ-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats.     

Selenihalanaerobacter shriftii gen. nov., sp. nov., a halophilic anaerobe from Dead Sea sediments that respires selenate

We isolated an obligately anaerobic halophilic bacterium from the Dead Sea that grew by respiration of selenate. The isolate, designated strain DSSe-1, was a gram-negative, non-motile rod. It oxidized glycerol or glucose to acetate + CO2 with concomitant reduction of selenate to selenite plus elemental selenium. Other electron acceptors that supported anaerobic growth on glycerol were nitrate and trimethylamine-N-oxide; nitrite, arsenate, fumarate, dimethylsulfoxide, thiosulfate, elemental sulfur, sulfite or sulfate could not serve as electron acceptors. Growth on glycerol in the presence of nitrate occurred over a salinity range from 100 to 240 g/l, with an optimum at 210 g/l. Analysis of the 16S rRNA gene sequence suggests that strain DSSe-1 belongs to the order Halanaerobiales, an order of halophilic anaerobes with a fermentative or homoacetogenic metabolism, in which anaerobic respiratory metabolism has never been documented. The highest 16S rRNA sequence similarity (90%) was found with Acetohalobium arabaticum (X89077). On the basis of physiological properties as well as the relatively low homology of 16S rRNA from strain DSSe-1 with known genera, classification in a new genus within the order Halanaerobiales, family Halobacteroidaceae is warranted. We propose the name Selenihalanaerobacter shriftii. Type strain is strain DSSe-1 (ATCC accession number BAA-73).     

Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy

Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAV(UK)DeltaS2 derived from a virulent proviral clone, EIAV(UK)DeltaS2/DU containing a second gene mutation in the virulent proviral clone, and EIAV(PR)DeltaS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 10(2) to 10(3) RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAV(PV) strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity.     

Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont

Corals and other cnidarians house photosynthetic dinoflagellate symbionts within membrane-bound compartments inside gastrodermal cells. Nutritional interchanges between the partners produce carbohydrates and lipids for metabolism, growth, energy stores, and cellular structures. Although lipids play a central role in the both the energetics and the structural/morphological features of the symbiosis, previous research has primarily focused on the fatty acid and neutral lipid composition of the host and symbiont. In this study we conducted a mass spectrometry-based survey of the lipidomic changes associated with symbiosis in the sea anemone Aiptasia pallida, an important model system for coral symbiosis. Lipid extracts from A. pallida in and out of symbiosis with its symbiont Symbiodinium were prepared and analyzed using negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry. Through this analysis we have identified, by exact mass and collision-induced dissociation mass spectrometry (MS/MS), several classes of glycerophospholipids in A. pallida. Several molecular species of di-acyl phosphatidylinositol and phosphatidylserine as well as 1-alkyl, 2-acyl phosphatidylethanolamine (PE) and phosphatidycholine were identified. The 1-alkyl, 2-acyl PEs are acid sensitive suggestive that they are plasmalogen PEs possessing a double bond at the 1-position of the alkyl linked chain. In addition, we identified several molecular species of phosphonosphingolipids called ceramide aminoethylphosphonates in anemone lipid extracts by the release of a characteristic negative product ion at m/z 124.014 during MS/MS analysis. Sulfoquinovosyldiacylglycerol (SQDG), an anionic lipid often found in photosynthetic organisms, was identified as a prominent component of Symbiodinium lipid extracts. A comparison of anemone lipid profiles revealed a subset of lipids that show dramatic differences in abundance when anemones are in the symbiotic state as compared to the non-symbiotic state. The data generated in this analysis will serve as a resource to further investigate the role of lipids in symbiosis between Symbiodinium and A. pallida.