AFM imaging of protein movements: histone H2A-H2B release during nucleosome remodeling

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi-Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi-Snf complexes.     

A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Recovery of human mesenchymal stem cells following dehydration and rehydration

As cell therapies advance from research laboratories to clinical application, there is the need to transport cells and tissues across long distances while maintaining cell viability and function. Currently cells are successfully stored and shipped under liquid nitrogen vapor. The ability to store these cells in the desiccated state at ambient temperature would provide tremendous economic and practical advantage. Human mesenchymal stem cells (hMSCs) have broad potential uses in tissue engineering and regeneration since they can differentiate along multiple lineages and support hematopoeisis. The current research applied recent technological advances in the dehydration and storage of human fibroblasts to hMSCs. Three conditions were tested: air-dried, air-dried and stored under vacuum (vacuum only), and incubated with 50 mM trehalose + 3% glycerol and then air-dried and stored under vacuum (vacuum + trehalose). Plates containing dehydrated hMSCs were shipped from San Diego to Baltimore overnight in separate FedEx cardboard boxes. The hMSCs were rehydrated with 3 ml of hMSC medium and were able to regain their spindle-shaped morphology and adhesive capability. In addition, they maintained high viability and proliferation capacity. Rehydrated and passaged cells continued to express the characteristic hMSC surface antigen panel. Additionally, cells showed constitutive levels of mRNA for a stromal factor and, when exposed to reagents known to induce differentiation, demonstrated upregulation of two tissue-specific messages indicative of differentiation potential for fat and bone. While our preliminary findings are encouraging, we still need to address consistency and duration of storage by considering factors such as cell water content, oxygen concentration, and the presence of free radicals.     

Observations on the postacrosomal region and the neck membranes of rabbit spermatozoa stained en bloc with uranyl acetate

Summary The postacrosomal region (PAC) of the head and the neck membranes of rabbit spermatozoa have been reinvestigated with the use of en bloc uranyl acetate staining. The fine structure of the PAC and neck membranes is visualized with great clarity and departs from that seen in rabbit sperm prepared by other methods. In cells so treated, the PAC contains a heavily enfolded nuclear envelope, a dense lamina attached to the plasmalemma by periodic connecting links and scattered dense material between the lamina and nuclear envelope. Evidence is presented that the dense lamina is a discrete structure, separated from the plasmalemma by the connecting links. The latter may be of a different composition from both the lamina and the plasmalemma. The lamina is a homogenous structure which resists degeneration under conditions which affect other components of the PAC. The membranes of the neck are a complex labyrinth of nuclear envelope, individual membranes, and membranes coursing through a matrical gound substance.This investigation was supported by Ford Foundation Grant 67–650.     

Zur Theorie der lateralen Inhibition

Summary Recently it was shown (Reichardt 1961) that lateral neural inhibition, such as was found in the lateral eye of Limulus polyphemus by Hartline and Ratliff, can in principle compensate for the dioptric apparatus of the eye. The model of lateral inhibition in Limulus developed there is here considered further, with emphasis on the changes in the effective structure of the nerve network (and the associated vector transformation) resulting from the forced inactivity of fibers whose inhibition exceeds their excitation. The stability of the network model as a function of the inhibition coefficients is studied and two theorems regarding the stability are proven. The dependence of the properties of the network on the pattern of receptor excitation are investigated and it is shown by examples that the network could be used for form discrimination. This model's relationship to previously known pattern recognition systems is discussed and its possible application in computer technology is mentioned.     

Phenotypic and Evolutionary Consequences of Social Behaviours: Interactions among Individuals Affect Direct Genetic Effects

Traditional quantitative genetics assumes that an individual''s phenotype is determined by both genetic and environmental factors. For many animals, part of the environment is social and provided by parents and other interacting partners. When expression of genes in social partners affects trait expression in a focal individual, indirect genetic effects occur. In this study, we explore the effects of indirect genetic effects on the magnitude and range of phenotypic values in a focal individual in a multi-member model analyzing three possible classes of interactions between individuals. We show that social interactions may not only cause indirect genetic effects but can also modify direct genetic effects. Furthermore, we demonstrate that both direct and indirect genetic effects substantially alter the range of phenotypic values, particularly when a focal trait can influence its own expression via interactions with traits in other individuals. We derive a function predicting the relative importance of direct versus indirect genetic effects. Our model reveals that both direct and indirect genetic effects can depend to a large extent on both group size and interaction strength, altering group mean phenotype and variance. This may lead to scenarios where between group variation is much higher than within group variation despite similar underlying genetic properties, potentially affecting the level of selection. Our analysis highlights key properties of indirect genetic effects with important consequences for trait evolution, the level of selection and potentially speciation.     

The evolution of endothermy in Cenozoic mammals: a plesiomorphic‐apomorphic continuum

The evolution of endothermy in birds and mammals was one of the most important events in the evolution of the vertebrates. Past tests of hypotheses on the evolution of endothermy in mammals have relied largely on analyses of the relationship between basal and maximum metabolic rate, and artificial selection experiments. I argue that components of existing hypotheses, as well as new hypotheses, can be tested using an alternative macrophysiological modeling approach by examining the development of endothermy during the Cenozoic. Recent mammals display a 10°C range in body temperature which is sufficiently large to identify the selective forces that have driven the development of endothermy from a plesiomorphic (ancestral) Cretaceous or Jurassic condition. A model is presented (the Plesiomorphic‐Apomorphic Endothermy Model, PAE Model) which proposes that heterothermy, i.e. bouts of normothermy (constant body temperature) interspersed with adaptive heterothermy (e.g. daily torpor and/or hibernation), was the ancestral condition from which apomorphic (derived), rigid homeothermy evolved. All terrestrial mammal lineages are examined for existing data to test the model, as well as for missing data that could be used to test the model. With the exception of Scandentia and Dermoptera, about which little is known, all mammalian orders that include small‐sized mammals (<500 g), have species which are heterothermic and display characteristics of endothermy which fall somewhere along a plesiomorphic‐apomorphic continuum. Orders which do not have heterothermic representatives (Cetartiodactyla, Perissodactyla, Pholidota, and Lagomorpha) are comprised of medium‐ to large‐sized mammals that have either lost the capacity for heterothermy, or in which heterothermy has yet to be measured. Mammalian heterothermy seems to be plesiomorphic and probably evolved once in the mammalian lineage. Several categories of endothermy are identified (protoendothermy, plesioendothermy, apoendothermy, basoendothermy, mesoendothermy, supraendothermy, and reversed mesoendothermy) to describe the evolution of endothermy during the Cenozoic. The PAE Model should facilitate the testing of hypotheses using a range of macrophysiological methods (e.g. the comparative method and the reconstruction of ancestral states).     

Disparities in Healthcare Utilisation Rates for Aboriginal and Non-Aboriginal Albertan Residents, 1997–2006: A Population Database Study

Background

It is widely recognised that significant discrepancies exist between the health of indigenous and non-indigenous populations. Whilst the reasons are incompletely defined, one potential cause is that indigenous communities do not access healthcare to the same extent. We investigated healthcare utilisation rates in the Canadian Aboriginal population to elucidate the contribution of this fundamental social determinant for health to such disparities.

Methods

Healthcare utilisation data over a nine-year period were analysed for a cohort of nearly two million individuals to determine the rates at which Aboriginal and non-Aboriginal populations utilised two specialties (Cardiology and Ophthalmology) in Alberta, Canada. Unadjusted and adjusted healthcare utilisation rates obtained by mixed linear and Poisson regressions, respectively, were compared amongst three population groups - federally registered Aboriginals, individuals receiving welfare, and other Albertans.

Results

Healthcare utilisation rates for Aboriginals were substantially lower than those of non-Aboriginals and welfare recipients at each time point and subspecialty studied [e.g. During 2005/06, unadjusted Cardiology utilisation rates were 0.28% (Aboriginal, n = 97,080), 0.93% (non-Aboriginal, n = 1,720,041) and 1.37% (Welfare, n = 52,514), p = <0.001]. The age distribution of the Aboriginal population was markedly different [2.7%≥65 years of age, non-Aboriginal 10.7%], and comparable utilisation rates were obtained after adjustment for fiscal year and estimated life expectancy [Cardiology: Incidence Rate Ratio 0.66, Ophthalmology: IRR 0.85].

Discussion

The analysis revealed that Aboriginal people utilised subspecialty healthcare at a consistently lower rate than either comparatively economically disadvantaged groups or the general population. Notably, the differences were relatively invariant between the major provincial centres and over a nine year period. Addressing the causes of these discrepancies is essential for reducing marked health disparities, and so improving the health of Aboriginal people.     

Genetic monitoring as a promising tool for conservation and management

In response to ever-increasing anthropogenic changes to natural ecosystems, regional, national and international organizations have established guidelines for monitoring biological diversity. Most monitoring programs, however, do not take full advantage of the potential afforded by molecular genetic markers, which can provide information relevant to both ecological and evolutionary time frames, while costing less and being more sensitive and reliable than traditional monitoring approaches. As several molecular and computational approaches are relatively new, many technical and theoretical issues remain to be resolved. Here, we illustrate how DNA and population genetic data can provide valuable information, often unattainable via other approaches, for monitoring species of management, conservation and ecological interest.