Determination of the complete order parameter tensor for a lipid methylene group from 1H- and 2H-NMR spin labels

Proton-proton dipolar splittings are obtained as a function of temperature for the α-methylene in a potassium palmitate - (β-ω) - d₂₉ (70 wt.%) / D₂O (30 wt.%) sample above and below the gel to liquid crystal phase transition. These splittings and corresponding deuteron quadrupole splittings are used to specify the complete order parameter tensor for the α-methylene group. Deduction of lipid structural information from the complete-order parameter tensor is discussed.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

The effect of gametic-phase disequilibrium on the prediction of response to recurrent selection in plants

Selection reduces additive genetic variation by generating gametic-phase disequilibrium, a phenomenon largely ignored when predicting response in plant breeding programs. The development of gametic-phase disequilibrium is here taken into account when predicting the response to selection for various schemes of recurrent selection applicable to plant populations. A general program permits prediction of response to selection from schemes of recurrent selection in which two or more rounds of selection occur in each cycle. An example from Sugar Beet, with alternate rounds of half-sib and S1 family selection, is illustrated. It is shown that failure to take into account the effects of gametic-phase disequilibrium can result in substantial overestimation of the response to selection as well as to changes in rank of the merits of alternative breeding schemes. For a given scheme, ignoring gametic-phase disequilibrium has only small effects on defining the optimum allocation of plots and the numbers of families tested.     

Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema

Background

Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding of endothelial cell function, their relevance to mechanisms in emphysema is questionable. Here we report a successful technique to isolate and characterise primary cultures of pulmonary microvascular endothelial cells from individuals with severe emphysema.

Methods

A lobe of emphysematous lung tissue removed at the time of lung transplantation surgery was obtained from 14 patients with severe end-stage disease. The pleura, large airways and large blood vessels were excised and contaminating macrophages and neutrophils flushed from the peripheral lung tissue before digestion with collagenase. Endothelial cells were purified from the cell mixture via selection with CD31 and UEA-1 magnetic beads and characterised by confocal microscopy and flow cytometry.

Results

Successful isolation was achieved from 10 (71%) of 14 emphysematous lungs. Endothelial cells exhibited a classical cobblestone morphology with high expression of endothelial cell markers (CD31) and low expression of mesenchymal markers (CD90, αSMA and fibronectin). E-selectin (CD62E) was inducible in a proportion of the endothelial cells following stimulation with TNFα, confirming that these cells were of microvascular origin.

Conclusions

Emphysematous lungs removed at the time of transplantation can yield large numbers of pulmonary microvasculature endothelial cells of high purity. These cells provide a valuable research tool to investigate cellular mechanisms in the pulmonary microvasculature relevant to the pathogenesis of emphysema.     

Self-Generated Chemoattractant Gradients: Attractant Depletion Extends the Range and Robustness of Chemotaxis

Chemotaxis is fundamentally important, but the sources of gradients in vivo are rarely well understood. Here, we analyse self-generated chemotaxis, in which cells respond to gradients they have made themselves by breaking down globally available attractants, using both computational simulations and experiments. We show that chemoattractant degradation creates steep local gradients. This leads to surprising results, in particular the existence of a leading population of cells that moves highly directionally, while cells behind this group are undirected. This leading cell population is denser than those following, especially at high attractant concentrations. The local gradient moves with the leading cells as they interact with their surroundings, giving directed movement that is unusually robust and can operate over long distances. Even when gradients are applied from external sources, attractant breakdown greatly changes cells'' responses and increases robustness. We also consider alternative mechanisms for directional decision-making and show that they do not predict the features of population migration we observe experimentally. Our findings provide useful diagnostics to allow identification of self-generated gradients and suggest that self-generated chemotaxis is unexpectedly universal in biology and medicine.     

Does stream flow structure woody riparian vegetation in subtropical catchments?

The primary objective of this study was to test the relevance of hydrological classification and class differences to the characteristics of woody riparian vegetation in a subtropical landscape in Queensland, Australia. We followed classification procedures of the environmental flow framework ELOHA – Ecological Limits of Hydrologic Alteration. Riparian surveys at 44 sites distributed across five flow classes recorded 191 woody riparian species and 15, 500 individuals. There were differences among flow classes for riparian species richness, total abundance, and abundance of regenerating native trees and shrubs. There were also significant class differences in the occurrence of three common tree species, and 21 indicator species (mostly native taxa) further distinguished the vegetation characteristics of each flow class. We investigated the influence of key drivers of riparian vegetation structure (climate, depth to water table, stream‐specific power, substrate type, degree of hydrologic alteration, and land use) on riparian vegetation. Patterns were explained largely by climate, particularly annual rainfall and temperature. Strong covarying drivers (hydrology and climate) prevented us from isolating the independent influences of these drivers on riparian assemblage structure. The prevalence of species considered typically rheophytic in some flow classes implies a more substantial role for flow in these classes but needs further testing. No relationships were found between land use and riparian vegetation composition and structure. This study demonstrates the relevance of flow classification to the structure of riparian vegetation in a subtropical landscape, and the influence of covarying drivers on riparian patterns. Management of environmental flows to influence riparian vegetation assemblages would likely have most potential in sites dominated by rheophytic species where hydrological influences override other controls. In contrast, where vegetation assemblages are dominated by a diverse array of typical rainforest species, and other factors including broad‐scale climatic gradients and topographic variables have greater influence than hydrology, riparian vegetation is likely to be less responsive to environmental flow management.