Habitat structure,conspecific presence and spatial variation in the recruitment of a temperate reef fish

Pronounced spatial variation in recruitment occurs in many marine invertebrate and fish populations and is thought to be critical to the demography of these species. In this study I examined the importance of habitat structure and the presence of conspecific residents to spatial variation in larval settlement and recruitment in a temperate fish Tautogolabrus adspersus. I define settlement as the movement of individuals from the water column to the benthic habitat, while I refer to recruitment as numbers of individuals surviving some arbitrary period of time after settlement. Experiments in which standard habitats were stocked with conspecifics showed that resident conspecifics were not an important factor contributing to small-scale variability in recruitment. Further correlative analyses demonstrated that large-scale variation in recruitment could not be explained by variability in older age classes. By contrast, manipulations of macroalgal structure within a kelp bed demonstrated that recruitment was significantly higher in habitats with a dense understory of foliose and filamentous algae than in habitats with only crustose algae. Understory algae varied in their pattern of disperison among sites, and the dispersion of fish matched that of the plants. In order to determine the effects of differences in patterns of algal dispersion on the demography of associated T. adspersus populations, I used experimental habitat units to manipulate patterns of dispersion. Settlement was significantly greater to randomly placed versus clumped habitats; however, no differences in recruitment between random and clumped habitats were detected. Because recruitment is a function of the numbers of settlers minus the subsequent loss of settlers, rates of mortality or migration must have been higher in the randomly placed habitats. These results are counter to the current paradigm for reef fishes which suggests that larval settlement is the crucial demographic process producing variability in population abundance. In this experiment patterns of settlement were modified by varying the patch structure of the habitat.Contribution number 278 from the Center for Marine Biology, University of New Hampshire     

Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelope

The recent solution of enteric bacterial porin structure, and new insights into the mechanism by which outer membrane receptor proteins recognize and internalize specific ligands, advocates the re-evaluation of TonB-dependent transport physiology. In this minireview we discuss the potential structural features of siderophore receptors and TonB, and use this analysis to evaluate both existing and new models of energy and signal transduction from the inner membrane to the outer membrane of gram-negative bacteria.     

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.     

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.     

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.     

Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain

Biologically active, mouse estrogen receptor hormone-binding domain (residues 313–599) overexpressed in Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [³H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [³H]estradiol was unaffected by Ca²⁺ and Mg²⁺ ions up to 1 mM but was significantly inhibited by Zn²⁺ ions at concentrations above 10 μM, an effect reversed by EDTA.     

Gene identification in a complex chromosomal continuum by local genomic cross-referencing

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.