Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.     

Humidity as a source of measurement error in osteometrics

A simple experiment was conducted to test the hypothesis that variation in humidity causes expansion of bone and, thereby, affects measurements of dried, preserved skulls. The experiment consisted of subjecting ten macaque skulls to increased humidity for 24 hours. Measurements of nine skull dimensions taken immediately before and after humidification revealed a statistically significant treatment effect of increased skull size with increased humidity. The length of the molar tooth row increased by about 0.1 mm (0.50%) while the greatest length of the skull increased by about 0.9 mm (0.57%). The specimens returned to their original dimensions within 1-2 days after being removed from the humidity chamber. These results confirm the impression gained by the practical experience of measuring museum specimens in different locations and environments. It appears that bony changes associated with humidity differences represent a real, though minor, source of measurement error in osteometrics.     

Restriction fragment analysis as a source of error in detection of heteroplasmic mtDNA mutations

The transition from A to G at nt 5656 (5656A→G) in mitochondrial DNA has been suggested to be a pathogenic mutation and, furthermore, a heteroplasmic one. We found that the mutation was present in 14 out of 83 healthy controls from northern Finland and that 5656A→G was exclusively associated with mtDNA haplogroup U. Interestingly, 5656A→G appeared to be heteroplasmic in NheI digestion of PCR fragments that were amplified by using a mismatched oligonucleotide primer creating a digestion site in the presence of the mutant variant. However, we did not detect the wild type genome in clones from such a sample and subsequent experiments revealed that the apparent heteroplasmy was due to inhibition of NheI by NaCl. Our results suggest that 5656A→G is a polymorphism and it may be highly characteristic for Finns. Furthermore, new heteroplasmic mutations identified by restriction fragment analysis should be adequately controlled for any false positive results that may be due to incomplete digestion.     

Suction sampling as a significant source of error in molecular analysis of predator diets

The molecular detection of predation is a fast growing field, allowing highly specific and sensitive detection of prey DNA within the gut contents or faeces of a predator. Like all molecular methods, this technique is prone to potential sources of error that can result in both false positive and false negative results. Here, we test the hypothesis that the use of suction samplers to collect predators from the field for later molecular analysis of predation will lead to high numbers of false positive results. We show that, contrary to previous published work, the use of suction samplers resulted in previously starved predators testing positive for aphid and collembolan DNA, either as a results of ectopic contamination or active predation in the collecting cup/bag. The contradictory evidence for false positive results, across different sampling protocols, sampling devices and different predator-prey systems, highlights the need for experimentation prior to mass field collections of predators to find techniques that minimise the risk of false positives.     

Evaluating mixed samples as a source of error in non-invasive genetic studies using microsatellites

The use of noninvasive genetic sampling (NGS) for surveying wild populations is increasing rapidly. Currently, only a limited number of studies have evaluated potential biases associated with NGS. This paper evaluates the potential errors associated with analysing mixed samples drawn from multiple animals. Most NGS studies assume that mixed samples will be identified and removed during the genotyping process. We evaluated this assumption by creating 128 mixed samples of extracted DNA from brown bear (Ursus arctos) hair samples. These mixed samples were genotyped and screened for errors at six microsatellite loci according to protocols consistent with those used in other NGS studies. Five mixed samples produced acceptable genotypes after the first screening. However, all mixed samples produced multiple alleles at one or more loci, amplified as only one of the source samples, or yielded inconsistent electropherograms by the final stage of the error-checking process. These processes could potentially reduce the number of individuals observed in NGS studies, but errors should be conservative within demographic estimates. Researchers should be aware of the potential for mixed samples and carefully design gel analysis criteria and error checking protocols to detect mixed samples.     

Structural changes in cellobiohydrolase I upon binding of a macromolecular ligand as evident by SAXS investigations

Xylan from Rhodymenia palmata binds to the cellobiohydrolase I from Trichoderma reesei (CBH I) or its core protein, inhibiting their activity. Adsorption onto microcrystalline cellulose (Avicel) is reduced approximately 30% for intact CBH I and nearly 50% for the core, whereas the effects with cellobiose are negligible. Structural changes concomitant with this binding are studied in solution by small angle X-ray scattering. In the "tadpole" structure typical for the CBH I [Abuja et al., 1988] the lengthening of the tail part is the most salient observation when xylan is present which accounts for an increase in Dmax (18.0 to 22.0 nm) and radius of gyration (4.74 to 5.18 nm). When xylan binds to the core the radius of gyration remains nearly unchanged. Here a model can be constructed showing a xylan molecule on the surface of the core protein near the tail part.     

Structural investigation of loose connective tissue by using a series of dextran fractions as non-interacting macromolecular probes.

The ability of the uncharged open-coil dextran molecules to penetrate tissue space, without coil-shape change, was utilized to probe (by partitioning experiments) the structural arrangement of the collagen-fibre network and the proteoglycan system. Hyaluronidase digests most of the proteoglycans away and enables the respective contributions to the exclusion volume to be evaluated by using a series of different-molecular-weight dextrans. It appears that the major part of the exclusion volume is due to the collagen-fibril as a rod and the dextran coil as an impenetrable sphere. The additional exclusion due to the proteoglycans could be accounted for by a set of points (regions of high proteoglycan-segment density) over which the dextran coild cannot pass. These points are an average of 50 nm apart and are indicative of local extensive entanglement of high-molecular-weight proteoglycans with each other. Reasons are given why these entanglements could not act as cross-links in long-term elastic loading of the tissue.     

An examination of observer bias as a source of error in surveys of seagrass shoots

Abstract Mensurative experiments investigated the effects of different observers on estimates of the density of shoots of two species of seagrass: Posidonia australis Hook and Zostera capricorni Aschers. Balanced programmes of sampling were used to examine variation in counts of seagrass shoots attributable to different observers, sizes of quadrats, depths and locations within large beds of each species of seagrass. A separate experiment examined differences between novice observers and a more experienced observer, when an ‘optimal’ size of sampling unit was used. Estimated densities of Zostera shoots varied inconsistently among observers, quadrats, depths and locations. Differences between observers were not affected by the size of quadrat used to count Posidonia shoots, but varied between locations in the seagrass bed. Experience had only a minor impact on biases. Only two of 12 novices produced counts that were different from the experienced observer. These results emphasize the importance of considering both accuracy and precision in the design of field studies of seagrasses.     

Competitive labeling as an approach to defining the binding surfaces of proteins: binding of monomeric insulin to lipid bilayers

The free monomeric form of insulin is known to adsorb strongly to many different surfaces. A question of physiological relevance for which no previous studies have been reported is whether the monomeric form of insulin binds to lipid bilayers. In order to answer this question, it is necessary to carry out studies at the very dilute concentrations (less than 10(-6) M) necessary to obtain this species. We have approached this problem by applying the method of competitive labeling [Hefford, M.A., Evans, R.M., Oda, G., & Kaplan, H. (1985) Biochemistry 24, 867-874] to study insulin at concentrations as low as 3 X 10(-8) M, in the presence and absence of large unilamellar liposomes. With 1-fluoro-2,4-dinitrobenzene as the labeling reagent, the relative chemical reactivities of the functional groups of insulin were found to decrease markedly when insulin was incubated with liposomes consisting of egg lecithin and cholesterol (2:1 mol/mol) in 1.0 M KCl, pH 7.5 at 37 degrees C. The decrease for each functional group was found to directly correlate with its proximity to the dimer-forming surface of the monomer. It is concluded that insulin binds to lipid bilayers in a specific orientation, with the dimer-forming surface interacting with the bilayer. These results demonstrate the feasibility of applying competitive labeling to obtain structure-function relationships of membrane-interactive proteins in general and monomeric insulin in particular.     

Kinetic analysis of the attachment of a biological particle to a surface by macromolecular binding

Motivated by the problem of microbial deposition, a dynamic model is developed for the attachment of a Brownian particle to a surface mediated by colloidal forces as well as macromolecular bridging. The model predicts the attachment probability of the particle to the surface based upon the free energy as a function of fluctuating bond number and separation distance from the surface. From this model, the mean first-passage time approach is used to predict the mean time required for the particle moving from the unattached state to the attached state based on the properties of the binding macromolecules. This approach provides an analytical approximation for mean transition time from the secondary energy minimum as well as the attachment rate constant for the general case where neither binding nor particle diffusion are necessarily rate-limiting.     

Restriction fragment analysis as a source of error in detection of heteroplasmic mtDNA mutations.

The transition from A to G at nt 5656 (5656A-->G) in mitochondrial DNA has been suggested to be a pathogenic mutation and, furthermore, a heteroplasmic one. We found that the mutation was present in 14 out of 83 healthy controls from northern Finland and that 5656A-->G was exclusively associated with mtDNA haplogroup U. Interestingly, 5656A-->G appeared to be heteroplasmic in NheI digestion of PCR fragments that were amplified by using a mismatched oligonucleotide primer creating a digestion site in the presence of the mutant variant. However, we did not detect the wild type genome in clones from such a sample and subsequent experiments revealed that the apparent heteroplasmy was due to inhibition of NheI by NaCl. Our results suggest that 5656A-->G is a polymorphism and it may be highly characteristic for Finns. Furthermore, new heteroplasmic mutations identified by restriction fragment analysis should be adequately controlled for any false positive results that may be due to incomplete digestion.     

Aminoglycoside binding sites in Escherichia coli as revealed by neomycin-gold labeling

A cytochemical technique for demonstration of neomycin binding sites by electron microscopy was developed and applied to Escherichia coli. Neomycin was conjugated chemically with bovine serum albumin (BSA). Colloidal gold was coated with the conjugated neomycin-BSA. The neomycin-BSA-gold was applied to thin sections of Epon-embedded E. coli and examined. Gold particles were observed on the outer membrane and the cytoplasmic membrane of E. coli. It was probably the ribosomes that were being labeled in the cytoplasm. Different cytochemical controls, including a number of inhibition tests and the use of BSA-gold, proved the specificity of this cytochemical technique and provided the biochemical significance of the observations.