Isolation and characterization of Campylobacter bacteriophages from retail poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4 degrees C.     

Bacterial community structure and location in Stilton cheese

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.     

Interaction of mannose-binding protein with associated serine proteases: effects of naturally occurring mutations

Mannose-binding protein (MBP; mannose-binding lectin) forms part of the innate immune system. By binding directly to carbohydrates on the surfaces of potential microbial pathogens, MBP and MBP-associated serine proteases (MASPs) can replace antibodies and complement components C1q, C1r, and C1s of the classical complement pathway. In order to investigate the mechanisms of MASP activation by MBP, the cDNAs of rat MASP-1 and -2 have been isolated, and portions encompassing the N-terminal CUB and epidermal growth factor-like domains have been expressed and purified. Biophysical characterization of the purified proteins indicates that each truncated MASP is a Ca(2+)-independent homodimer in solution, in which the interacting modules include the N-terminal two domains. Binding studies reveal that both MASPs associate independently with rat MBP in a Ca(2+)-dependent manner through interactions involving the N-terminal three domains. The biophysical properties of the truncated MASPs indicate that the interactions with MBP leading to complement activation differ significantly from those between components C1q, C1r, and C1s of the classical pathway. Analysis of MASP binding by rat MBP containing naturally occurring mutations equivalent to those associated with human immunodeficiency indicates that binding to both truncated MASP-1 and MASP-2 proteins is defective in such mutants.     

Use of bicuculline, a GABA antagonist, as a template for the development of a new class of ligands showing positive allosteric modulation of the GABA(A) receptor

Analogues of bicuculline devoid of the benzo ring fused to the lactone moiety were prepared by reacting 2-(tert-butyl-dimethylsiloxy)furans with 3,4-dihydroisoquinolinium salts. Some of these compounds (e.g., ROD185, 8) acted as modulators of the GABAA receptor, displacing ligands of the benzodiazepine binding site. They also strongly stimulated GABA currents mediated by recombinant GABA(A) receptors expressed in Xenopus oocytes.     

Photoinhibition in differently coloured juvenile leaves of Syzygium species

Photoinhibition, as measured by the dark-adapted chlorophyll a fluorescence ratio Fv/Fm, was assessed in Syzygium moorei, a species with dark green juvenile leaves, Syzygium corynanthum, which has light green juvenile leaves, and two species with pink-red juvenile leaves (Syzygium wilsonii and Syzygium luehmannii). All plants were glasshouse-grown (maximum PPFD 1500 mol m^-2 s^-1) under optimum nutrition and water.When measured at midday, dark-adapted Fv/Fm ratios of juvenile leaves gradually increased in all species as percentage of full leaf expansion (% FLE) increased. Fluorescence measurement 3h after sunset or pre-dawn also showed a developmental effect on Fv/Fm, with juvenile leaves of S. luehmannii and S. wilsonii showing much lower Fv/Fm at all stages of development. Dark-adapted Fv/Fm, values in both juvenile and mature leaves generally never exceeded 0.8 at any stage in any of the species.Courses of Fv/Fm on sunny days showed greater diurnal photoinhibition in green juvenile (c. 50% FLE) leaves of S. moorei (24%) and S. corynanthum (36%) than in mature leaves of the previous flush in these species (<10%). Diurnal photoinhibition was statistically similar (18-24%) in pink-red juvenile and green mature leaves of S. luehmannii and S. wilsonii. Re-positioning juvenile leaves of S. wilsonii horizontally increased diurnal photoinhibition.Exposure of leaves to a standard mild photoinhibitory light treatment (30 min at 1000 mol m^-2s^-1) showed that juvenile leaves of all species had a lower percentage of high energy state quenching (qE) and a higher percentage of photoinhibitory quenching (qI) than mature leaves.     

A comparison of the development and metabolic activity of mycorrhizas formed by arbuscular mycorrhizal fungi from different genera on two tropical forage legumes

 Two glasshouse experiments were done to assess the development and metabolic activity of mycorrhizas formed by isolates of arbuscular mycorrhizal fungi (AMF) from three different genera, Acaulospora, Gigaspora and Glomus on either Pueraria phaseoloides L. or Desmodium ovalifolium L. plants. The second of the two experiments included three levels of a localised phosphate source in the pots. Alkaline phosphatase (ALP), stained histochemically in the intra-radical mycelium (IRM) of AMF over sequential harvests, did not provide a direct marker for the efficiency of AMF in mobilising phosphorus (P) for plant growth and development. The ability of the extra-radical mycelium (ERM) to scavenge a localised phosphate source, determined by its extraction from buried 35-μm mesh pouches, was dependent on the species of AMF tested. This work indicates that AMF from different genera have unique patterns of mycelial development when forming mycorrhizas with tropical hosts in the presence of a localised phosphate source. AMF also appear to have different mechanisms for the control of P transfer, within the mycelium, to the host. The significance of the architecture of the ERM is discussed as well as the localisation of ALP in the IRM in determining the efficiency of AMF in terms of P accumulation in planta and subsequent growth of plants. Accepted: 19 August 1998     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998