Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.     

Parasitoid diets: Does superparasitism pay?

The ability of insect parasitoids to discriminate between parasitized and unparasitized hosts is well documented. Despite this, hosts that have been parasitized more than once are frequently found, an occurrence known as superparasitism. Since superparasitism results in interlarval competition, it was generally assumed to be caused by discriminatory 'mistakes'. Recently, theoretical studies have suggested that under certain circumstances superparasitism can be optimal. Superparasitism can thus be viewed as an active foraging strategy rather than as a passive process. Because parasitoids show a direct link between foraging success and reproductive output, they afford important opportunities to test evolutionary models of behaviour.     

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G-banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable "marker" chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated from LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to demonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellular morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.     

Methods for recovering poliovirus and rotavirus from oysters.

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.     

Factors affecting the distribution of stream macroinvertebrates in geothermal areas: Taupo Volcanic Zone,New Zealand

The distribution of macroinvertebrates was investigated among sites within five geothermally influenced and two non- or minimally-influenced streams in the Taupo Volcanic Zone, New Zealand, to examine the responses of communities to broad environmental gradients within and among habitats. To date, examination of geothermal stream macroinvertebrates has typically been from single habitats, and has not been examined over a regional scale. Sites within and among streams represented a range of sizes, depths, water velocities and substrate types. Sites with little or no geothermal influence typically had temperatures less than 15°C and pH between 5 and 8. Four of the geothermally influenced streams had temperatures greater than 25°C, and ranged from alkaline (pH 9.1) to highly acidic (pH 3.0). Most taxa recorded were typical inhabitants of non-geothermal streams that are tolerant to elevated temperatures, extreme pH conditions and/or high toxicant levels. Diptera, Coleoptera and Mollusca dominated geothermal sites, and Ephemeroptera, Plecoptera and Trichoptera were absent. In addition, an obligate dweller of geothermal habitats, Ephydrella thermarum, and a nonindigenous tropical gastropod, Melanoides tuberculata, were recorded. Canonical correspondence analysis implicated multiple factors in determining the distribution of invertebrates over the region. Overall, distribution was most strongly associated with temperature gradients, particularly longitudinally within streams. This distribution was likely directly related to species temperature tolerances, but also temperature effects on algal abundance and composition, its differential effects on species’ potential competitors and predators, and its effects on toxicant availability. Differences in invertebrate composition among streams were associated with major differences in pH and substrate. Increased acidity was associated with a significant decrease in invertebrate taxa richness, with acidic sites having a limited fauna dominated by dipterans (e.g., Naonella sp., Polypedilum sp.). Stability of flow and environmental conditions may enhance competitive interactions among taxa, enhancing the importance of substrate type in these systems. The presence of non-indigenous species (e.g., M. tuberculata, Poecilia reticulata), currently limited in distribution, also affected species composition. Overall, communities become less speciose, although more unusual in composition, with increased geothermal influence. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: K. Martens     

Isolation and characterisation of 14-S RNA from spinach chloroplasts.

14-S RNA was purified from spinach chloroplasts. It has a molecular weight of 0.43 . 10(6) and the following nucleotide composition: 20% CMP, 23.9% AMP, 24.2% GMP and 31.9% UMP. The accumulation of 14-S RNA in chloroplasts of cotyledons of dark-grown plants is stimulated by light. Conditions are described for the isolation of 14-S RNA in the absence of appreciable fragmentation of chloroplast 23-S rRNA and the evidence that it represents a distinct type of chloroplast RNA is discussed. Translation of 14-S RNA in a protein synthesising system from Escherichia coli gives rise to two polypeptides with molecular weights of 13 200 and 12 600 and the possible role of 14-S RNA as a chloroplast messenger is discussed.     

The importance of bead size measurement in mass-transfer modelling with immobilised cells

The effective diffusivity of O₂ inside immobilised cell particles has been much discussed. Most reported estimates are based on fitting a mass-transfer reaction model to measured total oxygen uptake rates. The particle diameter has the largest single influence in such models, but its accurate measurement has probably recieved insufficient attention. We have studied sorbitol and glucose oxidation by cells of Gluconobacter suboxydans entrapped in calcium alginate gel beads. These beads were found to shrink rapidly in air, so that size measurement under water is essential. By comparison with rigid particles of similar known size, it was shown that measurement of the microscopic image gives a systematic underestimate. In consequence, the fitted oxygen diffusivity will be around 20% too low. Careful attention to size measurement gave good agreement between diffusivity estimates from beads with different mean sizes and cell loadings, with a best value of 2.51 × 10⁹ m²s^–1, 92% of the value for pure water. The estimated diffusivity is not significantly affected by a distribution of bead sizes with up to 10% standard deviation about the same mean.     

XE7: a novel splicing factor that interacts with ASF/SF2 and ZNF265

Pre-mRNA splicing is performed by the spliceosome. SR proteins in this macromolecular complex are essential for both constitutive and alternative splicing. By using the SR-related protein ZNF265 as bait in a yeast two-hybrid screen, we pulled out the uncharacterized human protein XE7, which is encoded by a pseudoautosomal gene. XE7 had been identified in a large-scale proteomic analysis of the human spliceosome. It consists of two different isoforms produced by alternative splicing. The arginine/serine (RS)-rich region in the larger of these suggests a role in mRNA processing. Herein we show for the first time that XE7 is an alternative splicing regulator. XE7 interacts with ZNF265, as well as with the essential SR protein ASF/SF2. The RS-rich region of XE7 dictates both interactions. We show that XE7 localizes in the nucleus of human cells, where it colocalizes with both ZNF265 and ASF/SF2, as well as with other SR proteins, in speckles. We also demonstrate that XE7 influences alternative splice site selection of pre-mRNAs from CD44, Tra2-β1 and SRp20 minigenes. We have thus shown that the spliceosomal component XE7 resembles an SR-related splicing protein, and can influence alternative splicing.     

Differential regulation of oestrogen receptor β isoforms by 5' untranslated regions in cancer

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs - ERβ- are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5' untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5'UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5'UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.     

Influence of ingrowth regions on bone remodelling around a cementless hip resurfacing femoral implant

Hip resurfacing arthroplasty is an alternative to traditional hip replacement that can conserve proximal bone stock and has gained popularity but bone resorption may limit implant survival and remains a clinical concern. The goal of this study was to analyze bone remodelling patterns around an uncemented resurfacing implant and the influence of ingrowth regions on resorption. A computed tomography-derived finite element model of a proximal femur with a virtually implanted resurfacing component was simulated under peak walking loads. Bone ingrowth was simulated by six interface conditions: fully bonded; fully friction; bonded cap with friction stem; a small bonded region at the stem-cup intersection with the remaining surface friction; fully frictional, except for a bonded band along the distal end of the cap and superior half of the cap bonded with the rest frictional. Interface condition had a large influence on remodelling patterns. Bone resorption was minimized when no ingrowth occurred at the bone-implant interface. Bonding only the superior half of the cap increased bone resorption slightly but allowed for a large ingrowth region to improve secondary stability.