A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Effect of intravenous infusion of insulin in diabetics with acute myocardial infarction.

Diabetes mellitus is associated with a high mortality after myocardial infarction. To see whether this may be decreased by improved diabetic control the effect of an insulin infusion regimen was studied in patients with acute myocardial infarction. From April 1982 to April 1983, 33 diabetics were admitted with acute myocardial infarction. Those being treated with diet alone or oral hypoglycaemic drugs continued with this unless control was poor, when they were changed to a "sliding scale" regimen of subcutaneous insulin injections thrice daily. Those already receiving insulin were maintained on thrice daily subcutaneous injections. From April 1983 to April 1984, 29 diabetics had acute myocardial infarction. Those receiving treatment with oral hypoglycaemic drugs or insulin were changed to continuous intravenous infusion of insulin, the aim being to maintain the blood glucose concentration at 4-7 mmol/I (72-126 mg/100 ml). Those being treated with diet alone continued with this if blood glucose concentrations were acceptable. Total mortality fell from 42% in the first year to 17% in the second (p less than 0.05). Over the same period mortality among non-diabetic patients with myocardial infarction did not change significantly. There was a significant fall in cardiac arrhythmias (expressed as the percentage of patients in whom arrhythmias were recorded) from 42% to 17% (p less than 0.05). The most significant fall in the incidence of complications occurred in those who had been receiving oral hypoglycaemic drugs on entry to the study (87% to 50%, p less than 0.05).     

Estrogen replacement stimulates fatty acid oxidation and impairs post-ischemic recovery of hearts from ovariectomized female rats

Women less than 50 years of age, the majority of whom are likely premenopausal and exposed to estrogen, are at greater risk of a poor short-term recovery after myocardial ischemia than men and older women. Since estrogen enhances non-cardiac lipid utilization and increased lipid utilization is associated with poor post-ischemic heart function, we determined the effect of estrogen replacement on post-ischemic myocardial function and fatty acid oxidation. Female Sprague-Dawley rats, either intact (n = 15) or ovariectomized and treated with 17beta-estradiol (0.1 mg x kg(-1) x day(-1), s.c., n = 14) or corn oil vehicle (n = 16) for 5 weeks, were compared. Function and fatty acid oxidation of isolated working hearts perfused with 1.2 mM [9,10-3H]palmitate, 5.5 mM glucose, 0.5 mM lactate, and 100 mU/L insulin were measured before and after global no-flow ischemia. Only 36% of hearts from estrogen-treated rats recovered after ischemia compared with 56% from vehicle-treated rats (p > 0.05, not significant), while 93% of hearts from intact rats recovered (p < 0.05). Relative to pre-ischemic values, post-ischemic function of estrogen-treated hearts (26.3 +/- 10.1%) was significantly lower than vehicle-treated hearts (53.4 +/- 11.8%, p < 0.05) and hearts from intact rats (81.9 +/- 7.0%, p < 0.05). Following ischemia, fatty acid oxidation was greater in estrogen-treated hearts than in the other groups. Thus, estrogen replacement stimulates fatty acid oxidation and impairs post-ischemic recovery of isolated working hearts from ovariectomized female rats.     

Synthesis and anti-staphylococcal activity of novel bacterial topoisomerase inhibitors with a 5-amino-1,3-dioxane linker moiety

Novel bacterial type II topoisomerase inhibitors (NBTIs) constitute a promising new class of antibacterial agents. We report a series of NBTIs with potent anti-staphylococcal activity and diminished hERG inhibition. Dioxane-linked compound 9 demonstrated MICs ≤1?μg/mL against both methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA), accompanied by reduced hERG inhibition as compared to cyclohexane- or piperidine-linked analogs.     

Physiological dynamics,reproduction‐maintenance allocations,and life history evolution

Allocation of resources to competing processes of growth, maintenance, or reproduction is arguably a key process driving the physiology of life history trade‐offs and has been shown to affect immune defenses, the evolution of aging, and the evolutionary ecology of offspring quality. Here, we develop a framework to investigate the evolutionary consequences of physiological dynamics by developing theory linking reproductive cell dynamics and components of fitness associated with costly resource allocation decisions to broader life history consequences. We scale these reproductive cell allocation decisions to population‐level survival and fecundity using a life history approach and explore the effects of investment in reproduction or tissue‐specific repair (somatic or reproductive) on the force of selection, reproductive effort, and resource allocation decisions. At the cellular level, we show that investment in protecting reproductive cells increases fitness when reproductive cell maturation rate is high or reproductive cell death is high. At the population level, life history fitness measures show that cellular protection increases reproductive value by differential investment in somatic or reproductive cells and the optimal allocation of resources to reproduction is moulded by this level of investment. Our model provides a framework to understand the evolutionary consequences of physiological processes underlying trade‐offs and highlights the insights to be gained from considering fitness at multiple levels, from cell dynamics through to population growth.     

The ancestor of the <Emphasis Type="Italic">Paulinella</Emphasis> chromatophore obtained a carboxysomal operon by horizontal gene transfer from a <Emphasis Type="Italic">Nitrococcus</Emphasis>-like γ-proteobacterium

Background  

Paulinella chromatophora is a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin that are closely related to free-living Prochlorococcus and Synechococcus species (PS-clade). Members of the PS-clade of cyanobacteria contain a proteobacterial form 1A RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) that was acquired by horizontal gene transfer (HGT) of a carboxysomal operon. In rDNA-phylogenies, the Paulinella chromatophore diverged basal to the PS-clade, raising the question whether the HGT occurred before or after the split of the chromatophore ancestor.     

Multiple-antibiotic resistance of Enterococcus spp. isolated from commercial poultry production environments

The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern. Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments. Of the 541 isolates recovered, Enterococcus faecalis (53%) and E. faecium (31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species. The prevalence of resistance among isolates of E. faecalis was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of E. faecium were observed to be more frequently resistant to fluoroquinolones and penicillins. Notably, 63% of the E. faecium isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the E. faecalis population, of which 7% of the isolates were resistant. The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine. The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production.     

Cytochrome c3 Mutants of Desulfovibrio desulfuricans

To explore the physiological role of tetraheme cytochrome c₃ in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c₃ in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.     

Evaluation and integration of 49 genome-wide experiments and the prediction of previously unknown obesity-related genes

MOTIVATION: Genome-wide experiments only rarely show resounding success in yielding genes associated with complex polygenic disorders. We evaluate 49 obesity-related genome-wide experiments with publicly available findings including microarray, genetics, proteomics and gene knock-down from human, mouse, rat and worm, in terms of their ability to rediscover a comprehensive set of genes previously found to be causally associated or having variants associated with obesity. RESULTS: Individual experiments show poor predictive ability for rediscovering known obesity-associated genes. We show that intersecting the results of experiments significantly improves the sensitivity, specificity and precision of the prediction of obesity-associated genes. We create an integrative model that statistically significantly outperforms all 49 individual genome-wide experiments. We find that genes known to be associated with obesity are significantly implicated in more obesity-related experiments and use this to provide a list of genes that we predict to have the highest likelihood of association for obesity. The approach described here can include any number and type of genome-wide experiments and might be useful for other complex polygenic disorders as well.     

Tolerance of Hordeum marinum accessions to O2 deficiency, salinity and these stresses combined

Background and Aims

When root-zone O₂ deficiency occurs together with salinity, regulation of shoot ion concentrations is compromised even more than under salinity alone. Tolerance was evaluated amongst 34 accessions of Hordeum marinum, a wild species in the Triticeae, to combined salinity and root-zone O₂ deficiency. Interest in H. marinum arises from the potential to use it as a donor for abiotic stress tolerance into wheat.

Methods

Two batches of 17 H. marinum accessions, from (1) the Nordic Gene Bank and (2) the wheat belt of Western Australia, were exposed to 0·2 or 200 mol m⁻³ NaCl in aerated or stagnant nutrient solution for 28–29 d. Wheat (Triticum aestivum) was included as a sensitive check species. Growth, root porosity, root radial O₂ loss (ROL) and leaf ion (Na⁺, K⁺, Cl⁻) concentrations were determined.

Key Results

Owing to space constraints, this report is focused mainly on the accessions from the Nordic Gene Bank. The 17 accessions varied in tolerance; relative growth rate was reduced by 2–38 % in stagnant solution, by 8–42 % in saline solution (aerated) and by 39–71 % in stagnant plus saline treatment. When in stagnant solution, porosity of adventitious roots was 24–33 %; salinity decreased the root porosity in some accessions, but had no effect in others. Roots grown in stagnant solution formed a barrier to ROL, but variation existed amongst accessions in apparent barrier ‘strength’. Leaf Na⁺ concentration was 142–692 µmol g⁻¹ d. wt for plants in saline solution (aerated), and only increased to 247–748 µmol g⁻¹ d. wt in the stagnant plus saline treatment. Leaf Cl⁻ also showed only small effects of stagnant plus saline treatment, compared with saline alone. In comparison with H. marinum, wheat was more adversely affected by each stress alone, and particularly when combined; growth reductions were greater, adventitious root porosity was 21 %, it lacked a barrier to ROL, leaf K⁺ declined to lower levels, and leaf Na⁺ and Cl⁻ concentrations were 3·1–9-fold and 2·8–6-fold higher, respectively, in wheat.

Conclusions

Stagnant treatment plus salinity reduced growth more than salinity alone, or stagnant alone, but some accessions of H. marinum were still relatively tolerant of these combined stresses, maintaining Na⁺ and Cl⁻ ‘exclusion’ even in an O₂-deficient, saline rooting medium.Key words: Aerenchyma, combined salinity and waterlogging, leaf Cl⁻, leaf K⁺, leaf Na⁺, radial O₂ loss, salt tolerance, salinity–waterlogging interaction, sea barleygrass, waterlogging tolerance, wheat, wild Triticeae