Visualization of birch pollen allergens using IgE-containing sera from human atopic individuals in immunogold labelling experiments

Summary Pollen from birch trees (Betula pendula) was fixed in paraformaldehyde with or without the addition of 0.5% cetylpyridinium chloride, dehydrated and embedded in Lowicryl K4M in the cold. Ultrathin sections were incubated using the following sequence of antibodies and antisera: IgE-containing serum from an atopic human individual allergic to birch pollen allergens, rabbit anti-human IgE antibodies, and colloidal gold-labelled goat anti-rabbit antibodies. Controls were performed by replacing the specific human antiserum by serum from an atopic person with a similar level of IgE antibodies directed against allergens other than birch pollen allergens, or by omitting the human antiserum or the anti-IgE antibody or both. In test experiments, there was a dense specific labelling of the exine and the cytoplasmic matrix of the pollen grain. There was moderate labelling of the apertural regions (poral plugs). There was no labelling of the intine. In pollen grains fixed with the addition of cetylpyridinium chloride, an electron-dense surface coat was precipitated on the outside of the pollen wall. This surface material also remained completely unlabelled.     

Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells

Quantitative immunoelectron microscopy often involves determining the distributions of gold label in different intracellular compartments and then drawing comparisons between compartments in the same sample of cells or between experimental groups of cells. In the case of within-group comparisons, recent developments in the estimation of relative labelling index and labelling density make it possible to test whether or not particular compartments are preferentially labelled. These methods are ideally suited to analysing gold label restricted to volume (organelle) or surface (membrane) compartments but may be modified to analyse label localised in mixtures of both. Here, a simple and efficient approach to drawing between-group comparisons for label associated with organelles and/or membranes is presented. The method relies on multistage random sampling of specimens (via blocks and microscopic fields) followed by simply counting gold particles associated with different compartments. The distributions of raw gold counts in different groups are then compared by contingency table analysis with statistical degrees of freedom for chi-squared values being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values then identify where the main between-group differences reside. The method requires no information about compartment size (for example, organelle profile area or membrane trace length) and does not even depend critically on standardising between-group magnification. Its application is illustrated using datasets from immunolabelling studies designed to localise the KDEL receptor, phosphatidyl-inositol 4,5-bisphosphate, GLUT4 and rab4 at the electron microscopic level.     

Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying)

In quantitative immunoelectron microscopy, subcellular compartments that are preferentially labelled with colloidal gold particles can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto, this approach has been limited to compartments which are either surface occupying (membranes) or volume occupying (organelles) but not a mixture of both (membranes and organelles). However, some antigens are known to translocate between membrane and organelle compartments and the problem then arises of expressing gold particle LDs in a consistent manner (e.g., as number per compartment profile area). Here, we present one possible solution to tackle this problem. With this method, each membrane is treated as a volume-occupying compartment and this is achieved by creating an acceptance zone at a fixed distance on each side of membrane images. Gold signal intensity is then expressed as an LD within the membrane profile area so created and this LD can be compared to LDs found in volume-occupying compartments. Acceptance zone width is determined largely by the expected dispersion of gold labelling. In some cases, the zone can be applied to all visible membrane images but there is a potential problem when image loss occurs due to the fact that membranes are not cut orthogonal to their surface but are tilted within the section. The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs). The fraction of membrane images forming LVWs can be estimated in two ways: goniometrically (by determining the angle at which images become unclear) or stereologically (by counting intersections with test lines). The fraction obtained by either method can then be used to calculate a factor correcting for membrane image loss. In turn, this factor is used to estimate the total gold labelling associated with the acceptance zone of the entire (volume-occupying) membrane. However calculated, the LDs over the chosen (membrane and organelle) compartments are used to obtain observed and expected gold particle counts. The observed distribution is determined simply by counting gold particles associated with each compartment. Next, an expected distribution is created by randomly superimposing test points and counting those hitting each compartment. LDs of the chosen compartments are used to calculate RLI and chi-squared values and these serve to identify those compartments in which there is preferential labelling. The methods are illustrated by synthetic and real data.     

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

Update on estimation of mutation rates using data from fluctuation experiments

This note discusses a minor mathematical error and a problematic mathematical assumption in Luria and Delbrück's (1943) classic article on fluctuation analysis. In addition to suggesting remedial measures, the note provides information on the latest development of techniques for estimating mutation rates using data from fluctuation experiments.     

Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals

Using serum from human atopic individuals with a sufficiently high titre of IgE and IgG antibodies to birch- or hazel-pollen allergens and antigens, the localization of IgE binding sites in birch- and hazel-pollen grains was determined by pre- and post-embedding electron microscopic immunoautoradiography with 125I-anti-IgE, whereas the IgG binding sites were localized in ultrathin sections of birch-pollen grains by the protein-A/gold technique. Concerning the distribution patterns of both IgE/IgG binding sites within the pollen grains, no difference could be observed in the dormant pollen grain: Labelling was found in the exine part of the pollen wall and throughout the highly condensed cytoplasm except for starch grains and lipid droplets. The intine part and the germination pores were almost completely unlabelled. In pollen grains which had been soaked in a hypotonic buffer for 15 min, however, IgE binding sites were predominantly localized within the intine and the germination pores. The specificity of the labelling reactions and the observed differences in the localization patterns are discussed.     

A model for the analysis of growth data from designed experiments

A model for growth data from designed experiments is presented which extends the stochastic differential equation of Sandland and McGilchrist (1979, Biometrics 35, 255-272). Residual maximum likelihood (REML) is used to estimate the parameters of the model. The model is easily extended to incomplete data and is shown to overcome some of the practical difficulties encountered with the profile model. The procedure is applied to data from experiments on pigs and sheep.     

On the evaluation of data from flow-dialysis experiments

Flow dialysis can be used to measure (i) ligand binding to macromolecules and (ii) the size of transmembrane ion gradients. Generally an approximate method is used to calculate the binding or gradient parameters from the raw data. Here we present a simple but exact method and evaluate the errors that may arise when the approximate method is used to calculate the magnitude of ion gradients. In addition, equations are presented that allow for a correction for sampling from or additions to the upper compartment of a flow-dialysis vessel during the measurements. Setty and Hendler [(1982) J. Biochem. Biophys. Methods 7, 35-46] have reported artifacts in the measurement of ion-gradients caused by the addition of electron donors to the upper compartment of a flow-dialysis cell. Here we extend their observations and suggest additional methods to prevent such artifacts.     

A new approach to estimating the pool of stable organic matter in soil using data from long-term field experiments

It is a necessity to have a successful method to separate, quantify and define the active and passive soil organic matter pools for appropriate verification of models. In this study, the organic carbon content of long-term bare fallow soils was used as an indicator of the size of the stable soil organic matter pool. Although soil texture and soil structure are widely accepted as having an influence on the stable pool, most soil organic models neglect the relationship between soil structure and carbon stabilization. Therefore, the aim of this presentation is to estimate the size of the stable carbon pool and to relate it to soil texture and structure properties. It was calculated that over 50 yr, under bare fallow conditions, the relative decrease in the amount of carbon (C) for the most stable pools ranged between 2 and 12%. In comparison, for the less stabilized pools the relative decrease was calculated from 50 to 100%. This indicates that the organic carbon content of long-term bare fallow soils should be very similar to the size of the most stable C pool. We also observed that the amounts of carbon associated with primary particles <20 μm for numerous soils with contrasting carbon content, soil texture, and management practices showed a lower and an upper limit. Both these limits and the carbon content of long-term bare fallow soils (which were assumed to be similar to the size of the stable pool) were related to the content of primary particles <20 μm in the soil. To calculate these relationships, an equation was used including one term to describe the influence of soil texture and another to describe that of soil structure. The calculated regression for the bare fallow soils corresponded very well to the lower limit of carbon content associated with primary particles <20 μm. The upper limit was estimated only by increasing the regression parameter which is related to the amount of C per unit primary particles <20 μm. Considering the many published results of the influence of soil texture and structure on carbon stabilization processes in soil, the stable pool may be defined as the capacity of soils to sorb C. The upper limit of carbon content associated with primary particles <20 μm may be interpreted as the capacity of soil to protect C. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

Topological model of the division process of cellular and subcellular compartments

The application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for a formation of a topological model of membrane transformations during the division process of cellular and subcellular compartments, has been shown. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicle. The analysis shows eight succeeding topological stages of membrane transformations during the division process and these stages are characterised. It is concluded that there is a vectorial translocation of lipid molecules from the inner layer of the membrane bounding the vesicle before the division process to the outer layer of the membranes after the division process and there is no lipid translocation from the outer layer to the inner layers during the division process.     

Topological analysis of the fusion process between cellular and subcellular compartments

The study presents an application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for topological analysis of membrane transformations during the fusion process between cellular and subcellular compartments. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicles. The analysis shows eight succeeding topological stages of membrane transformations during the fusion process and these stages are characterized. It is concluded that there is a vectorial translocation of lipid molecules from the outer layers of the membranes before the fusion process to the internal layer of the membrane bounding the vesicle after the fusion process and there is no lipid translocation in the reverse direction.     

Estimating and comparing the distribution of onset of disease from prevalence case-control data

Age at ascertainment from prevalence case-control data identifies the age-specific odds of disease. When age at onset is available from the cases, the conditional distribution of age at onset, given that disease occurs, is identifiable. Combining both kinds of information by introducing a multiplicative intercept allows identification of the marginal distribution of age at onset. Here, the approach is extended to the two-sample setting through a generalization of the multiplicative intercept model. The efficiency of the approach is explored and a test statistic based on the integrated difference between distribution function estimates is proposed. An approach to regularization of the likelihood is discussed. The methods are illustrated through an application to data on colorectal polyps obtained from a case-control study of individuals undergoing colonoscopy.     

Estimating heritabilities and genetic correlations: comparing the 'animal model' with parent-offspring regression using data from a natural population

Quantitative genetic parameters are nowadays more frequently estimated with restricted maximum likelihood using the 'animal model' than with traditional methods such as parent-offspring regressions. These methods have however rarely been evaluated using equivalent data sets. We compare heritabilities and genetic correlations from animal model and parent-offspring analyses, respectively, using data on eight morphological traits in the great reed warbler (Acrocephalus arundinaceus). Animal models were run using either mean trait values or individual repeated measurements to be able to separate between effects of including more extended pedigree information and effects of replicated sampling from the same individuals. We show that the inclusion of more pedigree information by the use of mean traits animal models had limited effect on the standard error and magnitude of heritabilities. In contrast, the use of repeated measures animal model generally had a positive effect on the sampling accuracy and resulted in lower heritabilities; the latter due to lower additive variance and higher phenotypic variance. For most trait combinations, both animal model methods gave genetic correlations that were lower than the parent-offspring estimates, whereas the standard errors were lower only for the mean traits animal model. We conclude that differences in heritabilities between the animal model and parent-offspring regressions were mostly due to the inclusion of individual replicates to the animal model rather than the inclusion of more extended pedigree information. Genetic correlations were, on the other hand, primarily affected by the inclusion of more pedigree information. This study is to our knowledge the most comprehensive empirical evaluation of the performance of the animal model in relation to parent-offspring regressions in a wild population. Our conclusions should be valuable for reconciliation of data obtained in earlier studies as well as for future meta-analyses utilizing estimates from both traditional methods and the animal model.     

A computer method for analysis of radioactivity data from single and double labeled experiments

A generally applicable program has been devised for the machine processing and plotting of radioactivity data. The program can be used with single or double labeled experiments; it includes numerous options which increase its flexibility and facilitate optimization of data presentation.     

Pooling and comparing estimates from several experiments of a Michaelis constant for an enzyme

Often the Michaelis constant of an enzyme will be determined several times. This may be done for various reasons such as ensuring reproducibility, comparing different enzyme preparations, or examining the effects of variations in experimental conditions. In these circumstances, two questions arise. First, how can the various estimates of the Michaelis constant be compared to determine whether they are the same within the limits of experimental variation? Secondly, if they are all the same, how can the values be combined to give an overall estimate? These questions are addressed here and a solution proposed in which the sets of data are pooled and analyzed with a separate maximum velocity for each set but a common Michaelis constant. The pooled data are partitioned in suitable ways and reanalyzed to examine, by means of a variance ratio test, whether a single Michaelis constant gives a satisfactory fit to the data.     

A graphical method to determine the ordering of moult,illustrated with data from the Blackshouldered Kite Elanus caeruleus

When the order in which feathers moult is not constant and the pattern is complex, it is difficult to discover the usual pattern simply by inspecting the data. This paper illustrates a method, the Gower analysis, that produces a graphical plot from which it is relatively easy to deduce the usual ordering.     

Determining the most important cellular characteristics for fracture healing using design of experiments methods

Computational models are employed as tools to investigate possible mechanoregulation pathways for tissue differentiation and bone healing. However, current models do not account for the uncertainty in input parameters, and often include assumptions about parameter values that are not yet established. The objective of this study was to determine the most important cellular characteristics of a mechanoregulatory model describing both cell phenotype-specific and mechanobiological processes that are active during bone healing using a statistical approach. The computational model included an adaptive two-dimensional finite element model of a fractured long bone. Three different outcome criteria were quantified: (1) ability to predict sequential healing events, (2) amount of bone formation at early, mid and late stages of healing and (3) the total time until complete healing. For the statistical analysis, first a resolution IV fractional factorial design (L₆₄) was used to identify the most significant factors. Thereafter, a three-level Taguchi orthogonal array (L₂₇) was employed to study the curvature (non-linearity) of the 10 identified most important parameters. The results show that the ability of the model to predict the sequences of normal fracture healing was predominantly influenced by the rate of matrix production of bone, followed by cartilage degradation (replacement). The amount of bone formation at early stages was solely dependent on matrix production of bone and the proliferation rate of osteoblasts. However, the amount of bone formation at mid and late phases had the rate of matrix production of cartilage as the most influential parameter. The time to complete healing was primarily dependent on the rate of cartilage degradation during endochondral ossification, followed by the rate of cartilage formation. The analyses of the curvature revealed a linear response for parameters related to bone, where higher rates of formation were more beneficial to healing. In contrast, parameters related to fibrous tissue and cartilage showed optimum levels. Some fibrous connective tissue- and cartilage formation was beneficial to bone healing, but too much of either tissue delayed bone formation. The identified significant parameters and processes are further confirmed by in vivo animal experiments in the literature. This study illustrates the potential of design of experiments methods for evaluating computational mechanobiological model parameters and suggests that further experiments should preferably focus at establishing values of parameters related to cartilage formation and degradation.     

A review comparing deoxyribonucleoside triphosphate (dNTP) concentrations in the mitochondrial and cytoplasmic compartments of normal and transformed cells

The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10(-7)) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm.     

Origin of terrestrial polypeptides: A theory based on data from discharge-tube experiments

Polymeric materials derived from HCN have been synthesized from reactants containing only carbon, hydrogen and nitrogen, as the solid product formed at high temperature on the walls of a discharge tube, and at room temperature from the gaseous products of that discharge condensed in a cold trap and allowed to warm up in the dark. These compounds were hydrolyzed with acid, and when possible with alkali. Amphoteric molecules were separated from the hydrolysate and examined for amino acids by GLC, after preparation of the TAB derivative. In all cases where nitrogenous solids were hydrolyzed, many natural and a few synthetic amino acids were formed, while blank runs indicated no trace of amino acids under the same treatment. These results have been used to show that many previous experiments in which amino acids have been synthesized from supposed ‘prebiological’ atmospheres have probably all involved the same general reaction steps.

1. Formation of HCN from high-energy reaction of C?H?N(?O) systems.
2. Radical or ionic polymerization or oligomerization of HCN.
3. Hydrolysis of the product.

A new theory for the origin of proteins on the primitive earth has been described in the light of these experimental results. It is suggested that high molecular weight aliphatic and aromatic hydro carbons and polymers derived from HCN are formed in the methane-ammonia atmosphere, and settle on the Earth's surface before the formation of oceans. Rainstorms wash the materials into alkaline pools, where the polymer is hydrolyzed in the presence of other materials necessary for the formation of replicating polypeptides (aromatic hydrocarbons, purines and pyrimidines and possibly phosphate ion). The close similarity between the products of discharge experiments and organic materials found in carbonaceous chondrites has also been discussed.     

Proteotoxic stress and ageing triggers the loss of redox homeostasis across cellular compartments

The cellular proteostasis network integrates the protein folding and clearance machineries in multiple sub‐cellular compartments of the eukaryotic cell. The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. A distinctive feature of the ER is its tightly controlled redox homeostasis necessary for the formation of inter‐ and intra‐molecular disulphide bonds. Employing genetically encoded in vivo sensors reporting on the redox state in an organelle‐specific manner, we show in the nematode Caenorhabditis elegans that the redox state of the ER is subject to profound changes during worm lifetime. In young animals, the ER is oxidizing and this shifts towards reducing conditions during ageing, whereas in the cytosol the redox state becomes more oxidizing with age. Likewise, the redox state in the cytosol and the ER change in an opposing manner in response to proteotoxic challenges in C. elegans and in HeLa cells revealing conservation of redox homeostasis. Moreover, we show that organelle redox homeostasis is regulated across tissues within C. elegans providing a new measure for organismal fitness.