Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay

Susan Maye is in receipt of a Teagasc Walsh Fellowship. Financial support by the Department of Agriculture, Food and the Marine is gratefully acknowledged.Copyright © Proprietors of Journal of Dairy Research 2015 (Institute of Food Research and the Hannah Research Institute)peer-reviewedWhile the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6·20 and 6·06 log CFU/ml, for raw bovine and human milks, respectively – the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.Department of Agriculture, Food and the Marin

Sand Body Geometry of the Wax Lake Outlet Delta Atchafalaya Bay, Louisiana

Deltas forming in Atchafalaya Bay, L-ouisiana, are the result of delta switching by the Mississippi River. The larger Lower Atchafalaya River delta has been heavily manipulated by dredging for navigation, but the Wax Lake Outlet delta is largely undisturbed and an excellent example of a \u27bayhead\u27 delta. Combining stratigraphy, aerial photography and digital terrain model data sets, the developmental history of this delta is presented. The Wax Lake Outlet delta is comprised of a typical upward-coarsening sequence, although its prodelta unit is extremely limited. Its plan-view form is typical of deltas developing in low-energy, unstratified, shallow basins. Early developmental processes were identified by Roberts and van Heerden (1992) Development through the 1980s involved the maturation of distributary channels. From 1989 to 1994, the majority of sediment was retained seaward of the delta proper, due to the efficiency of the distributary’ system. Greatest sand body thicknesses were found on the upstream portions of delta lobes, but not necessarily at points of bifurcation. Estimates of sand body volume range from 129 to 139 x 106 m3. A small area of the Atchafalaya River delta investigated for comparison also contains an upward-coarsening sequence but with upper and lower coarsegrained bounding units generated by dredging activity. Comparison of the Wax Lake Outlet delta to other Mississippi deltas reveals some similar processes of development despite differences in settings. The Wax Lake Outlet delta has shown a lower rate of infilling compared to subdeltas of the Mississippi River Balize delta due to the relative immaturity of the Atchafalaya. Growth curves based on terrain model data predict an area of 111 km2 (at and above 0.0 NGVD) by the year 2000, which falls within the range of values given by the Wells et al. (1982) generic model based on the Mississippi subdeltas

Counter-Reformation: the Search for a Unified John Donne

The agenda of this paper is a re-examination of the de-facto formation of a literary character. John Donne is one of history’s most celebrated metaphysical poets. A quasi-contemporary of William Shakespeare, his biography has been similarly (if obviously to a lesser degree) plumbed. The turn of the 17th century offers, in terms of hard facts, only tantalizing details left by fortuitous accident, and it has been the realm of biographers and early modern scholars to piece together the fragments. In the case of John Donne, this has manifested as a genealogy of literary biography that frequently melds scant fact, poetic manuscripts, and agreed upon assumptions to give us an image of a man who is less person than personification of the tumultuous and revolutionary times in which he lived. The image formed of John Donne is a chronologically distinct collection of personalities: the scholar, the rogue, the soldier and the theologian, culminating in a dichotomy between the youthful Jack Donne, and the revered Dr. Donne. This paper will seek to trouble that construction in two ways. In the first part, the paper will examine three major literary biographies which helped to construct this image—John Donne: A Life,by R.C. Bald, John Donne: Life, Mind, and Art by John Carey, and Donne: The Reformed Soul, by John Stubbs—and deconstruct their conclusions. In the second part of the paper, I will address the poetic canon of John Donne. In the collusion of both I will attempt to propose a unified John Donne, replete with biographical and literary continuity

Measuring dry plant residues in grasslands: A case study using AVIRIS

Grasslands, savannah, and hardwood rangelands are critical ecosystems and sensitive to disturbance. Approximately 20 percent of the Earth's surface are grasslands and represent 3 million ha. in California alone. Developing a methodology for estimating disturbance and the effects of cumulative impacts on grasslands and rangelands is needed to effectively monitor these ecosystems. Estimating the dry biomass residue remaining on rangelands at the end of the growing season provides a basis for evaluating the effectiveness of land management practices. The residual biomass is indicative of the grazing pressure and provides a measure of the system capacity for nutrient cycling since it represents the maximum organic matter available for decomposition, and finally, provides a measure of the erosion potential for the ecosystem. Remote sensing presents a possible method for measuring dry residue. However, current satellites have had limited application due to the coarse spatial scales (relative to the patch dynamics) and insensitivity of the spectral coverage to resolve dry plant material. Several hypotheses for measuring the biochemical constituents of dry plant material, particularly cellulose and lignin, using high spectral resolution sensors were proposed. The use of Airborne Visible/Infrared Imaging Spectrometers (AVIRIS) to measure dry plant residues over an oak savannah on the eastern slopes of the Coast Range in central California was investigated and it was asked what spatial and spectral resolutions are needed to quantitatively measure dry plant biomass in this ecosystem

Deletional Analysis of the rod Photoreceptor Cell Peripherin/RDS Carboxy-Terminal Region

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311-322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of -19, -29, -39 and -59 amino acids were designed to disrupt the α-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76 kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19 amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion. © 2002 Elsevier Science Ltd

Deletional Analysis of the Rod Photoreceptor Cell Peripherin/RDS Carboxy-Terminal Region

The C-terminal region of peripherin/rds contains three predicted α-helical domains. One of these domains, corresponding to amino acids 311–322, form an amphiphilic α-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional α-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Δ1, Δ2, Δ3 and Δ4), corresponding to deletions of −19, −29, −39 and −59 amino acids were designed to disrupt the α-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76 kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Δ1, Δ2 and Δ3 mutants formed homo-oligomeric complexes, while the Δ4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19 amino acids, a region downstream of the fusion peptide that is deleted in the Δ1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/ rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion

Enhancing the stress responses of probiotics for a lifestyle from gut to product and back again

Before a probiotic bacterium can even begin to fulfill its biological role, it must survive a battery of environmental stresses imposed during food processing and passage through the gastrointestinal tract (GIT). Food processing stresses include extremes in temperature, as well as osmotic, oxidative and food matrix stresses. Passage through the GIT is a hazardous journey for any bacteria with deleterious lows in pH encountered in the stomach to the detergent-like properties of bile in the duodenum. However, bacteria are equipped with an array of defense mechanisms to counteract intracellular damage or to enhance the robustness of the cell to withstand lethal external environments. Understanding these mechanisms in probiotic bacteria and indeed other bacterial groups has resulted in the development of a molecular toolbox to augment the technological and gastrointestinal performance of probiotics. This has been greatly aided by studies which examine the global cellular responses to stress highlighting distinct regulatory networks and which also identify novel mechanisms used by cells to cope with hazardous environments. This review highlights the latest studies which have exploited the bacterial stress response with a view to producing next-generation probiotic cultures and highlights the significance of studies which view the global bacterial stress response from an integrative systems biology perspective

Labour commodification in the employment heartland: Union responses to teachers' temporary work

This article analyses the commodification of professional labour and union responses to these processes within the employment heartland. It explores the category of fixed-contract or ‘temporary’ employment using Australian public school teaching as the empirical lens. Established to address intensifying conditions of labour market insecurity, the union-led creation of the temporary category was intended to partly decommodify labour by providing intermediate security between permanent and ‘casual’ employment. However, using historical case and contemporary survey data, we discern that escalation of temporary teacher numbers and intensifying work-effort demands concurrently increased insecurity within the teacher workforce, constituting recommodification. The paper contributes to scant literature on unions and commodification, highlighting that within the current marketised context, labour commodification may occur through contradictory influences at multiple levels, and that union responses to combat this derogation of work must similarly be multi-level and sustained