A tapetal ablation transgene induces stable male sterility and slows field growth in Populus

The field performance of genetic containment technologies–considered important for certain uses of transgenic trees in forestry–is poorly known. We tested the efficiency of a barnase gene driven by the TA29 tapetum-dominant promoter for influencing growth rate and inducing male sterility in a field trial of transgenic hybrid poplar (Populus tremula?×?Populus tremuloides). When the growth of 18 transgenic insertion events with the sterility transgene were compared to non-transgenic controls after two growing seasons, they grew 40 % more slowly in stem volume, and all but one transgenic event grew significantly more slowly than the control. In contrast, when we compared the growth of transgenic trees containing four kinds of β-glucuronidase (GUS) reporter gene constructs to non-transgenic trees—all of which had been produced using the same transformation method and poplar clone and grown at the same field site—there were no statistically significant differences in growth after three growing seasons. In 2 years where gross pollen release from catkins was monitored and found to be abundant in the control, no pollen was visible in the transgenic trees; microscopy suggested the cause was tapetal collapse and revealed the presence of a very few normal-sized pollen grains of unknown viability. In two additional years when viable, well-formed pollen was microscopically documented in controls, and no pollen could be observed in any transgenic trees. We conclude that this construct resulted in robust and possibly complete male sterility that was stable over 4 years in the field.     

Second Generation Inactivated Eastern Equine Encephalitis Virus Vaccine Candidates Protect Mice against a Lethal Aerosol Challenge

Currently, there are no FDA-licensed vaccines or therapeutics for eastern equine encephalitis virus (EEEV) for human use. We recently developed several methods to inactivate CVEV1219, a chimeric live-attenuated eastern equine encephalitis virus (EEEV). Dosage and schedule studies were conducted to evaluate the immunogenicity and protective efficacy of three potential second-generation inactivated EEEV (iEEEV) vaccine candidates in mice: formalin-inactivated CVEV1219 (fCVEV1219), INA-inactivated CVEV1219 (iCVEV1219) and gamma-irradiated CVEV1219 (gCVEV1219). Both fCVEV1219 and gCVEV1219 provided partial to complete protection against an aerosol challenge when administered by different routes and schedules at various doses, while iCVEV1219 was unable to provide substantial protection against an aerosol challenge by any route, dose, or schedule tested. When evaluating antibody responses, neutralizing antibody, not virus specific IgG or IgA, was the best correlate of protection. The results of these studies suggest that both fCVEV1219 and gCVEV1219 should be evaluated further and considered for advancement as potential second-generation inactivated vaccine candidates for EEEV.     

Effect of pseudophosphorylation and cross-linking by lipid peroxidation and advanced glycation end product precursors on tau aggregation and filament formation

Accumulation of hyperphosphorylated Tau protein as paired helical filaments in pyramidal neurons is a major hallmark of Alzheimer disease. Besides hyperphosphorylation, other modifications of the Tau protein, such as cross-linking, are likely to contribute to the characteristic features of paired helical filaments, including their insolubility and resistance against proteolytic degradation. In this study, we have investigated whether the four reactive carbonyl compounds acrolein, malondialdehyde, glyoxal, and methylglyoxal accelerate the formation of Tau oligomers, thioflavin T-positive aggregates, and fibrils using wild-type and seven pseudophosphorylated mutant Tau proteins. Acrolein and methylglyoxal were the most reactive compounds followed by glyoxal and malondialdehyde in terms of formation of Tau dimers and higher molecular weight oligomers. Furthermore, acrolein and methylglyoxal induced the formation of thioflavin T-fluorescent aggregates in a triple pseudophosphorylation-mimicking mutant to a slightly higher degree than wild-type Tau. Analysis of the Tau aggregates by electron microscopy study showed that formation of fibrils using wild-type Tau and several Tau mutants could be observed with acrolein and methylglyoxal but not with glyoxal and malondialdehyde. Our results suggest that reactive carbonyl compounds, particularly methylglyoxal and acrolein, could accelerate tangle formation in vivo and that this process could be slightly accelerated, at least in the case of methylglyoxal and acrolein, by hyperphosphorylation. Interference with the formation or the reaction of these reactive carbonyl compounds could be a promising way of inhibiting tangle formation and neuronal dysfunction in Alzheimer disease and other tauopathies.     

Isolation and Characterization of Coumaphos-Metabolizing Bacteria from Cattle Dip

Coumaphos, an organophosphate insecticide, is used for tick control in cattle dipping vats along the U.S.-Mexican border. Recently, several vats (problem vats) have experienced a loss of efficacy because of microbial degradation. Three morphologically distinct bacteria (designated B-1, B-2, and B-3) that metabolized coumaphos were isolated from enrichment cultures that were initiated from problem vat dip material. In general, amino acids, pyrimidines, and acetate supported growth; carbohydrates were not utilized. Only B-2 required growth factors. In resting cell experiments, coumaphos was hydrolyzed to diethylthiophosphoric acid and chlorferon by all three isolates. Chlorferon was subsequently metabolized by B-1 and B-2 to α-chloro-β-methyl-2,3,4-trihydroxy-trans-cinnamic acid. Only B-1 produced additional metabolites. Experiments with [benzo ring-labeled U-¹⁴C]coumaphos or chlorferon demonstrated that B-1 was capable of both mineralizing and incorporating into biomass the aromatic portion of the molecule. The majority of label, however, was recovered in the form of soluble products, including α-chloro-β-methyl-2,3,4-trihydroxy-trans-cinnamic acid. Although B-1 had the capacity to use chlorferon as a carbon source at low concentrations (100 μg/ml), visible growth at higher concentrations (1,000 μg/ml) was not observed. The addition of 400 μg of chlorferon per ml to B-1 cells in the mid-log phase of growth resulted in complete inhibition of growth, while the addition of 100 to 200 μg of chlorferon per ml resulted in partial inhibition. The growth of B-2 and B-3 was inhibited by 100 μg of chlorferon per ml. These data suggest that, although B-1 and, to a lesser extent, B-2 and B-3 are responsible for the primary degradation of coumaphos, other organisms in the enrichment culture may play a secondary role in coumaphos degradation by removing inhibitory products of coumaphos metabolism.     

Release of Glutamate and Aspartate from CA1 Synaptosomes: Selective Modulation of Aspartate Release by Ionotropic Glutamate Receptor Ligands

Abstract: Synaptosomes prepared from area CA1 of the rat hippocampus were used to determine (a) whether Schaffer collateral-commissural-ipsilateral associational terminals release both aspartate and glutamate in a Ca²⁺-dependent manner when reuptake of released glutamate is minimal and (b) whether autoreceptor mechanisms described in CA1 or hippocampal slices could reflect direct actions of glutamate receptor ligands on the synaptic terminal. When challenged for 1 min with either 25 m M K⁺ or 300 µ M 4-aminopyridine, CA1 synaptosomes released both glutamate and aspartate in a Ca²⁺-dependent manner. The glutamate/aspartate ratio was ∼5:1 in each case. K⁺-evoked glutamate release was unaffected by ligands active at NMDA or ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Unlike glutamate release, the release of aspartate was enhanced by NMDA, and this effect was blocked by d -2-amino-5-phosphonovalerate ( d -AP5). Kainate selectively depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) selectively increased the K⁺-evoked release of aspartate. AMPA enhanced aspartate release, like the antagonist CNQX. When applied in the presence of diazoxide, which blocks the desensitization of AMPA receptors, AMPA and kainate both depressed aspartate release. These findings support the view that Schaffer collateral-commissural-ipsilateral associational terminals release aspartate as well as glutamate and that these two release processes are regulated by different autoreceptor mechanisms.     

Applications of transposition mutagenesis in antibiotic producing streptomycetes

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the no rmally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.     

Ultrastructural analysis of DNA complexes during transfection and intracellular transport.

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2-1-micro m aggregates composed of 30-50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.     

A germline clone screen for meiotic mutants in Drosophila melanogaster

Using an FLP/FRT-based method to create germline clones, we screened Drosophila chromosome arms 2L and 3R for new female meiotic mutants. The screen was designed to recover mutants with severe effects on meiotic exchange and/or segregation. This screen yielded 11 new mutants, including six alleles of previously known meiotic genes (c(2)M and ald/mps1). The remaining five mutants appear to define at least four new genes whose ablation results in severe meiotic defects. Three of the novel meiotic mutants were identified at the molecular level. Two of these, mcm5(A7) and trem(F9), define roles in meiotic recombination, while a third, cona(A12), is important for synaptonemal complex assembly. Surprisingly, five of the nine mutants for which the lesion has been identified at the molecular level are not the result of mutations characteristic of EMS mutagenesis, but rather due to the insertion of the transposable element Doc. This study demonstrates the utility of germline clone-based screens for the discovery of strong meiotic mutants, including mutations in essential genes, and the use of molecular genetic techniques to map the loci.