Determination of Total Protein Content of Bacterial Cells by SYPRO Staining and Flow Cytometry

An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of Bacteria (an Arcobacter sp., a Cytophaga sp., an Oceanospirillum sp., a Pseudoalteromonas sp., and a Vibrio sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell⁻¹ (r² = 0.93, n = 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell⁻¹.     

TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures in Escherichia coli

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402-410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coli allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery, traM, traB, traD, and traE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essential tra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.     

Differences in morphology and C/N-balance between clones ofPhragmites australis within a plantation at a degraded fen

The morphology, productivity and C/N-balance of 9 different clones ofPhragmites australis planted in 1997 in a degraded fen area of 40,000 m² was investigated in order to estimate the degree of variation between the genotypes. The planted reed clones showed significant differences in morphology, standing crop and stand structure at the same site. The above-ground biomass of some reed clones was due to high culms and large leaf areas, while among others it was due to high shoot densities and small culms. The productivity of the individual clones also differed. At the end of the 1998 growing season the standing crop of the clones ranged from about 700 to 2,000 g of dry matter per m². Differences were found in the C/N-dynamics as in the standing stock of total nitrogen in the above-ground biomass (ranging from 15 to 50 g N/m²) and the relative nitrogen content of the shoots. Furthermore, seasonal changes in the amount of free amino acids and carbohydrates in the basal internodes of the different clones were compared. The patterns are discussed with respect to the nutritional status of the reed plants. In conclusion, the results suggest high genotypic variation despite the comparable site conditions and thus a strong influence of genetically determined differences in growth and resource exploitation on the characteristics of reed clones.     

Tree Migration-Rates: Narrowing the Gap between Inferred Post-Glacial Rates and Projected Rates

Faster-than-expected post-glacial migration rates of trees have puzzled ecologists for a long time. In Europe, post-glacial migration is assumed to have started from the three southern European peninsulas (southern refugia), where large areas remained free of permafrost and ice at the peak of the last glaciation. However, increasing palaeobotanical evidence for the presence of isolated tree populations in more northerly microrefugia has started to change this perception. Here we use the Northern Eurasian Plant Macrofossil Database and palaeoecological literature to show that post-glacial migration rates for trees may have been substantially lower (60–260 m yr^–1) than those estimated by assuming migration from southern refugia only (115–550 m yr^–1), and that early-successional trees migrated faster than mid- and late-successional trees. Post-glacial migration rates are in good agreement with those recently projected for the future with a population dynamical forest succession and dispersal model, mainly for early-successional trees and under optimal conditions. Although migration estimates presented here may be conservative because of our assumption of uniform dispersal, tree migration-rates clearly need reconsideration. We suggest that small outlier populations may be a key factor in understanding past migration rates and in predicting potential future range-shifts. The importance of outlier populations in the past may have an analogy in the future, as many tree species have been planted beyond their natural ranges, with a more beneficial microclimate than their regional surroundings. Therefore, climate-change-induced range-shifts in the future might well be influenced by such microrefugia.     

Spatial Organization and Mechanical Properties of the Pericellular Matrix on Chondrocytes

A voluminous polymer coat adorns the surface of many eukaryotic cells. Although the pericellular matrix (PCM) often extends several microns from the cell surface, its macromolecular structure remains elusive. This massive cellular organelle negotiates the cell’s interaction with surrounding tissue, influencing important processes such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. Investigations of the PCM’s architecture and function have been hampered by the difficulty of visualizing this invisible hydrated structure without disrupting its integrity. In this work, we establish several assays to noninvasively measure the ultrastructure of the PCM. Optical force probe assays show that the PCM of rat chondrocyte joint (RCJ-P) cells easily reconfigures around optically manipulated microparticles, allowing the probes to penetrate into rather than compress the matrix. We report distinct changes in forces measured from PCMs treated with exogenous aggrecan, illustrating the assay’s potential to probe proteoglycan distribution. Measurements reveal an exponentially increasing osmotic force in the PCM arising from an inherent concentration gradient. With this result, we estimate the variation of the PCM’s mesh size (correlation length) to range from ∼100 nm at the surface to 500 nm at its periphery. Quantitative particle exclusion assays confirm this prediction and show that the PCM acts like a sieve. These assays provide a much-needed tool to study PCM ultrastructure and its poorly defined but important role in fundamental cellular processes.     

Widespread co‐occurrence of divergent mitochondrial haplotype lineages in a Central American species of poison frog (Oophaga pumilio)

Aim To analyse the phylogeographic structure of the strawberry poison frog, Oophaga pumilio (Dendrobatidae), across a large part of its range using a combination of mitochondrial and nuclear markers. Location Costa Rica and Panama. Methods Sequence analyses of a mitochondrial (cytochrome b) and a nuclear (RAG‐1) gene fragment as well as analyses of seven microsatellite loci were carried out on 269 individuals of O. pumilio sampled from 24 localities and on two individuals of O. vicentei. Results Two main mitochondrial haplotype lineages, corresponding to a northern (north Costa Rica) and a southern (south Costa Rica and eastern Panama) lineage, were identified. They differed by up to 7% uncorrected distance. We observed co‐occurrence of both lineages in seven populations in Costa Rica and Panama, indicating a pattern of extensive admixture. The two main mitochondrial lineages of O. pumilio roughly corresponded to a previously described phylogeographic pattern. Microsatellites indicate admixture spanning over a wide geographic area, but significant variation between the northern and southern groups was also found with microsatellite data. While microsatellite data reconstructed a separation south of an assumed Caribbean valley barrier, mitochondrial haplotypes of the ‘southern lineage’ shifted this barrier towards the north. Main conclusions Despite admixture, all three markers showed significant variation between the northern and southern groups. Phylogeographical breaks known from other anuran species in the study region could not be verified for O. pumilio. The unexpected clustering of the population from Escudo de Veraguas and the individuals of O. vincentei with the northern O. pumilio lineage indicates the need for a fundamental and careful taxonomic revision, including an interspecific phylogeography of the entire genus.     

Ethnicity- and Sex-Based Discrimination and the Maintenance of Self-Esteem

The psychological underpinnings of labor market discrimination were investigated by having participants from Israel, the West Bank and Germany (N = 205) act as employers in a stylized employment task in which they ranked, set wages, and imposed a minimum effort level on applicants. State self-esteem was measured before and after the employment task, in which applicant ethnicity and sex were salient. The applicants were real people and all behavior was monetarily incentivized. Supporting the full self-esteem hypothesis of the social identity approach, low self-esteem in women was associated with assigning higher wages to women than to men, and such behavior was related to the maintenance of self-esteem. The narrower hypothesis that successful intergroup discrimination serves to protect self-esteem received broader support. Across all participants, both ethnicity- and sex-based discrimination of out-groups were associated with the maintenance of self-esteem, with the former showing a stronger association than the latter.     

Diversity of coevolved weeds in smallholder maize fields of Mexico and Zimbabwe

Theory and empirical data suggest the areas of origin of a crop to be the general area of origin of its coevolved weeds. These longer evolved weeds would have an advantage over species with a shorter evolutionary time and migrate more successfully. We seek to identify patterns by comparing two regions with a shared crop, similar physiographic traits, but little direct contact, one of which is the area of origin of the crop. We compared the diversity of the maize weed flora and its edible components between two rural villages each of Oaxaca, Mexico, and Honde Valley, Zimbabwe, using vegetation sampling, interviews and participatory observation. The Mexican fields had higher species richness and diversity than the Zimbabwean ones. Species richness and densities were higher in the villages that receive more rainfall. Mexican fields had a mainly native weed flora with almost 80% American species and very few of African origin, whereas Zimbabwe had 32% of American and 50% of African origin. The regions shared seven American species and one of African origin. American/Mesoamerican agrestal weeds appear to be more successful in maize. Subsistence farmers in both study areas consumed about 19 edible weed species of which four were common to all villages. Our results also suggest that the presence of 3–4 species of edible weeds per field may be a general pattern in the maize-based systems, and that people not necessarily want or need more, so usefulness—at least as an edible plant—would have a limited influence on migration success.     

Toxicity of Functional Nano-Micro Zinc Oxide Tetrapods: Impact of Cell Culture Conditions,Cellular Age and Material Properties

With increasing production and applications of nanostructured zinc oxide, e.g., for biomedical and consumer products, the question of safety is getting more and more important. Different morphologies of zinc oxide structures have been synthesized and accordingly investigated. In this study, we have particularly focused on nano-micro ZnO tetrapods (ZnO-T), because their large scale fabrication has been made possible by a newly introduced flame transport synthesis approach which will probably lead to several new applications. Moreover, ZnO-T provide a completely different morphology then classical spherical ZnO nanoparticles. To get a better understanding of parameters that affect the interactions between ZnO-T and mammalian cells, and thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material properties on cytotoxicity. Our results demonstrate that the cell density of fibroblasts in culture along with their age, i.e., the number of preceding cell divisions, strongly affect the cytotoxic potency of ZnO-T. Concerning the material properties, the toxic potency of ZnO-T is found to be significantly lower than that of spherical ZnO nanoparticles. Furthermore, the morphology of the ZnO-T influenced cellular toxicity in contrast to surface charges modified by UV illumination or O₂ treatment and to the material age. Finally, we have observed that direct contact between tetrapods and cells increases their toxicity compared to transwell culture models which allow only an indirect effect via released zinc ions. The results reveal several parameters that can be of importance for the assessment of ZnO-T toxicity in cell cultures and for particle development.