Agrobacterium tumefaciens-mediated transformation of severalRubus genotypes and recovery of transformed plants

Experiments in shoot regeneration and virulentAgrobacterium tumefaciens-mediated transformation were used to develop a protocol forRubus transformation. This protocol was then used to produce transformedRubus plants fromin vitro internodes inoculated with anAgrobacterium tumefaciens encoding neomycin phosphotransferase on its disarmed T-DNA. Two transformed plants were selected from 800 inoculations on a medium containing 10 µg ml^–1 kanamycin. Results indicated that this level of kanamycin successfully selected against non-transformed cells but did not reduce the number of transformed, kanamycin-resistant, shoots formed. Enzyme assays and Southern blot analysis verified the presence of the -glucuronidase gene in the plant genome.     

Effects of Motility and Adsorption Rate Coefficient on Transport of Bacteria through Saturated Porous Media

Three strains of Pseudomonas fluorescens with different motility rates and adsorption rate coefficients were injected into porous-medium reactors packed with l-mm-diameter glass spheres. Cell breakthrough, time to peak concentration, tailing, and cell recovery were measured at three interstitial pore velocities (higher than, lower than, and much lower than the maximal bacterial motility rate). All experiments were done with distilled water to reduce the effects of growth and chemotaxis. Contrary to expectations, motility did not result in either early breakthrough or early time to peak concentration at flow velocities below the motility rate. Bacterial size exclusion effects were shown to affect breakthrough curve shape at the very low flow velocity, but no such effect was seen at the higher flow velocity. The tendency of bacteria to adsorb to porous-medium surfaces, as measured by adsorption rate coefficients, profoundly influenced transport characteristics. Cell recoveries were shown to be correlated with the ratio of advective to adsorptive transport in the reactors. Adsorption rate coefficients were found to be better predictors of microbial transport phenomena than individual characteristics, such as size, motility, or porous-medium hydrodynamics.     

Use of Fluorinated Compounds To Detect Aromatic Metabolites from m-Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol → 4-hydroxy-2-methylbenzoic acid → 4-hydroxybenzoic acid → benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol → 5-fluoro-4-hydroxy-2-methylbenzoic acid → 3-fluoro-4-hydroxybenzoic acid → 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-¹⁴C]cresol was recovered predominantly as [¹⁴C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols.     

A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations

p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay alows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.     

Interactive effects of rising temperatures and urbanisation on birds across different climate zones: A mechanistic perspective

Climate change and urbanisation are among the most pervasive and rapidly growing threats to biodiversity worldwide. However, their impacts are usually considered in isolation, and interactions are rarely examined. Predicting species' responses to the combined effects of climate change and urbanisation, therefore, represents a pressing challenge in global change biology. Birds are important model taxa for exploring the impacts of both climate change and urbanisation, and their behaviour and physiology have been well studied in urban and non-urban systems. This understanding should allow interactive effects of rising temperatures and urbanisation to be inferred, yet considerations of these interactions are almost entirely lacking from empirical research. Here, we synthesise our current understanding of the potential mechanisms that could affect how species respond to the combined effects of rising temperatures and urbanisation, with a focus on avian taxa. We discuss potential interactive effects to motivate future in-depth research on this critically important, yet overlooked, aspect of global change biology. Increased temperatures are a pronounced consequence of both urbanisation (through the urban heat island effect) and climate change. The biological impact of this warming in urban and non-urban systems will likely differ in magnitude and direction when interacting with other factors that typically vary between these habitats, such as resource availability (e.g. water, food and microsites) and pollution levels. Furthermore, the nature of such interactions may differ for cities situated in different climate types, for example, tropical, arid, temperate, continental and polar. Within this article, we highlight the potential for interactive effects of climate and urban drivers on the mechanistic responses of birds, identify knowledge gaps and propose promising future research avenues. A deeper understanding of the behavioural and physiological mechanisms mediating species' responses to urbanisation and rising temperatures will provide novel insights into ecology and evolution under global change and may help better predict future population responses.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Ingestion of Salmonella enterica Serotype Poona by a Free-Living Nematode, Caenorhabditis elegans, and Protection against Inactivation by Produce Sanitizers

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables. Caenorhabditis elegans was selected as a model to study the effectiveness of sanitizers in killing Salmonella enterica serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on S. enterica serotype Poona were treated with produce sanitizers. Treatment with 20 μg of free chlorine/ml significantly (α = 0.05) reduced the population of S. enterica serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of S. enterica serotype Poona cells surviving treatments with 20 to 500 μg of chlorine/ml, suggesting that reductions caused by treatment with 20 μg of chlorine/ml resulted from inactivation of S. enterica serotype Poona on the surface of C. elegans but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 μg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log₁₀ CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 μg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log₁₀ CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing S. enterica serotype Poona and lactic acid was the most effective. Treatment with up to 500 μg of chlorine/ml, 1% hydrogen peroxide, 2,550 μg of Sanova/ml, 40 μg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of C. elegans. Treatments were also applied to cantaloupe rind and lettuce inoculated with S. enterica serotype Poona or C. elegans that had ingested S. enterica serotype Poona. Protection of ingested S. enterica serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that S. enterica serotype Poona ingested by C. elegans may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.     

Nest site selection by yellow-faced honeyeaters Lichenostomus chrysops

When choosing subsequent nest sites, species that produce multiple broods in a single breeding season have the option of dispersing from a site where they previously suffered depredation, i.e., a predator-avoidance tactic. In our study on yellow-faced honeyeaters Lichenostomus chrysops it was found that 89% of nest failures were attributed to nest depredation, the primary cause of reproductive failure. Pairs re-nested further from depredated nesting attempts than from successful nesting attempts and progressively higher above the ground as the breeding season progressed. Pairs nesting in dioecious Coprosma quadrifida plants only nested in non-fruiting male plants. Artificial nests were used to test the hypothesis that nest height and plant preferences were strategies to reduce the risk of depredation. There was no evidence that either higher nests or nests in non-fruiting C. quadrifida achieved reduced levels of depredation during 14 days of artificial nest exposure. Specific nest site characteristics were not found to be associated with nest outcome for either natural or artificial nests. Our study provides further evidence that species may choose a diverse range of nest sites in order to avoid predators from developing specific search images and then, following depredation, compensate by rapidly re-nesting away from the failed attempt.     

Foraging,Delayed Maturation,and Advantages of Cooperative Breeding in White-winged Choughs,Corcorax melanorhamphos

White-winged choughs live in groups which cooperate in all aspects of rearing young, affording an opportunity to examine the influence of cooperation on foraging behavior. The amount of food choughs forage for themselves and feed to young increases with age, supporting the idea that individuals which dispersed to breed would have difficulty in rearing young. When feeding nestlings, individuals in the two larger groups returned to the nest less often and with larger loads than the individuals in the smallest group. Choughs in the smallest group also consumed less food at the beginning of each trip from the nest than those in the larger groups. We suggest that these measures indicate the greater efficiency allowed to individuals in larger groups when foraging from the nest. In all groups, individuals returning to the nest simultaneously with other group members carried smaller loads than those returning alone. We propose that returning in groups enables all nestlings of asynchronously hatched broods to obtain sufficient food.