Macromolecular changes caused by formalin fixation and antigen retrieval

Although the mechanics of formalin fixation and antigen retrieval have been studied extensively and reviewed periodically, little attention has been directed toward conformational changes in target molecules. Formaldehyde changes the shape of tissue molecules by appending small hydroxymethyl groups to them. These adducts, in turn, can react with other tissue molecules to form crosslinks, or they can participate in a variety of reactions during tissue processing, including formation of imines, ethoxymethyl adducts, and further crosslinks. Under the influence of alcohol dehydration, fixed DNA may fragment and form a variety of depurination products. The situation becomes even more complex with short fixation times because under these conditions, the dehydrating agent used for tissue processing denatures macromolecules in other ways, most notably through rearrangement of molecular shape to move hydrophobic realms outward and hydrophilic areas inward (hydrophobic inversions). How tissue molecules are modified affects the outcome of immunohistochemical staining and prospects for restoration of antigenicity. Immunoreacitivity may be compromised because epitopes are either sterically hidden, but otherwise unaffected, or they have been altered more directly. Enzyme-based retrieval methods are best suited for the former because they literally snip the molecule apart to reveal the portions of interest. Heat-induced retrieval with buffers can demodify affected epitopes by removing adducts and breaking crosslinks. The choice of temperature and pH is usually critical for optimal retrieval. Effective temperatures are directly related to the strength of bonds-higher temperatures are needed to break stronger bonds. The pH of the retrieval solution determines the charge on the tissue molecule; the goal is to create a charge that causes the demodified molecule to assume a near natural conformation. Rational use of these concepts should lead to better control of immunohistochemical reactions.     

Effect of shear on the inactivation kinetics of the enzyme dextransucrase

An inactivation model previously developed to characterize the rate of enzyme activity loss in unstirred solutions was extended to take into account orthokinetic interactions resulting from convective mixing. A synergistic relationship between shear rate and temperature was observed; the rate of inactivation of the enzyme dextransucrase was unaffected by the action of shear below 25 degrees C, but was increased by the shear rate at 30 degrees C. Shear rate does not appear to influence the equilibrium between native and denatured dextransucrase either directly in solution or indirectly by augmenting the turnover of the gas-liquid interface. However, a second-order plot of the inverse of relative activity (A(O)/A) versus Gt (shear rate x time) of dextransucrase at a constant temperature was linear because of the influence of shear on the coagulation of the denatured enzyme. The addition of 0.01 g L(-1) of polyethylene glycol (MW 20,000) blocked this coagulation reaction, thereby completely inhibiting the shear-induced inactivation of dextransucrase at 30 degrees C. (c) 1993 John Wiley & Sons, Inc.     

Carbon dioxide absorption characterization of a bioreactor for biomass production ofPhormidium bohneri: comparative study of three types of diffuser

Under both laboratory-controlled and outdoor conditions, mass culture ofPhormidium bohneri has shown low growth rates and filament aggregate or floc instability. In order to test for a possible carbonaceous supply limitation, a mass transfer characterization study of the reactor used was conducted. To examine different conditions of aeration, the overall volumetric mass transfer coefficient, K_La(C0₂), and the energy and physical transfer efficiency for CO₂ were determined for three air-flow rates and three types of diffuser. For the different aeration conditions studied, the reactor showed adequate mixing properties with respect to CO₂. However, under low air-flow rate conditions, even with the most efficient diffuser, the CO₂ transfer capacity appeared limiting. On the other hand, at high air-flow rates, floc breakage as a result of shear stress appeared to limit bioreactor efficiency.     

Dyes from a twenty-first century perspective

In June 2008, the Biological Stain Commission sponsored A Seminar on Dyes and Staining the purpose of which was twofold: first, to show that very useful information applicable to biomedical dyes and staining is available from unrelated disciplines and second, to summarize modern thinking on how dyes, solvents, and tissues interact to produce selective staining. In this introduction to the papers from the symposium, we acknowledge that biomedical dye research has declined as newer technologies have gained importance. We should point out, however, that dyes and staining still are vitally important. Moreover, needs abound for innovative studies concerned with dye analysis, synthesis, and mode of action. Concepts and tools from unrelated fields hold promise for significant breakthroughs in many areas of interest.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross- reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand - Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.      

Phylogenetic utility of elongation factor-1 alpha in noctuoidea (Insecta: Lepidoptera): the limits of synonymous substitution

To test its phylogenetic utility, nucleotide sequence variation in a 1,240-bp fragment of the elongation factor-1 alpha (EF-1 alpha) gene was examined in 49 moth species representing the major groups of the superfamily Noctuoidea. Both parsimony and distance analyses supported the monophyly of nearly all groups for which there are clear morphological synapomorphies. Clades of subfamily rank and lower, probably mid-Tertiary and younger, were strongly supported. The third codon position contains 88% of variable sites, and approaches saturation at approximately 20% sequence divergence, possibly due to among-site rate heterogeneity and composition bias; higher divergences occur only in association with shifts in composition. Surprisingly, the few nonsynonymous changes appear no more phylogenetically reliable than synonymous changes. Signal strength for basal divergences is weak and fails to improve with character weighting; thus, dense taxon sampling is probably needed for strong inference from EF-1 alpha regarding deeper splits in Noctuoidea (probably early Tertiary). EF-1 alpha synonymous changes show promise for phylogeny reconstruction within Noctuidae and other groups of Tertiary age.      

Fractional clearance of urate: validation of measurement in spot-urine samples in healthy subjects and gouty patients

Introduction

Hyperuricemia is the greatest risk factor for gout and is caused by an overproduction and/or inefficient renal clearance of urate. The fractional renal clearance of urate (FCU, renal clearance of urate/renal clearance of creatinine) has been proposed as a tool to identify subjects who manifest inefficient clearance of urate. The aim of the present studies was to validate the measurement of FCU by using spot-urine samples as a reliable indicator of the efficiency of the kidney to remove urate and to explore its distribution in healthy subjects and gouty patients.

Methods

Timed (spot, 2-hour, 4-hour, 6-hour, 12-hour, and 24-hour) urine collections were used to derive FCU in 12 healthy subjects. FCUs from spot-urine samples were then determined in 13 healthy subjects twice a day, repeated on 3 nonconsecutive days. The effect of allopurinol, probenecid, and the combination on FCU was explored in 11 healthy subjects. FCU was determined in 36 patients with gout being treated with allopurinol. The distribution of FCU was examined in 118 healthy subjects and compared with that from the 36 patients with gout.

Results

No substantive or statistically significant differences were observed between the FCUs derived from spot and 24-hour urine collections. Coefficients of variation (CVs) were both 28%. No significant variation in the spot FCU was obtained either within or between days, with mean intrasubject CV of 16.4%. FCU increased with probenecid (P < 0.05), whereas allopurinol did not change the FCU in healthy or gouty subjects. FCUs of patients with gout were lower than the FCUs of healthy subjects (4.8% versus 6.9%; P < 0.0001).

Conclusions

The present studies indicate that the spot-FCU is a convenient, valid, and reliable indicator of the efficiency of the kidney in removing urate from the blood and thus from tissues. Spot-FCU determinations may provide useful correlates in studies investigating molecular mechanisms underpinning the observed range of efficiencies of the kidneys in clearing urate from the blood.

Trial Registration

ACTRN12611000743965     

Human responses to propionic acid. I. Quantification of within- and between-participant variation in perception by normosmics and anosmics

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra- threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri- threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.