The male germ unit of Rhododendron : quantitative cytology, three-dimensional reconstruction, isolation and detection using fluorescent probes

The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.

Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Emerging engineered magnetic nanoparticulate probes for molecular MRI of atherosclerosis : how far have we come?

Atherosclerosis is a chronic, progressive, immunoinflammatory disease of the large and medium-sized arteries, and a major cause of cardiovascular diseases. Atherosclerosis often progresses silently for decades until the occurrence of a major catastrophic clinical event such as myocardial infarction, cardiac arrest and stroke. The main challenge in the diagnosis and management of atherosclerosis is to develop a safe, noninvasive technique that is accurate and reproducible, which can detect the biologically active high-risk vulnerable plaques (with ongoing active inflammation, angiogenesis and apoptosis) before the occurrence of an acute clinical event. This Journal Article reviews the events involved in the pathogenesis of atherosclerosis in light of recently advanced understanding of the molecular pathogenesis of the disease. Next, we elaborate on the interesting developments in molecular MRI, by describing the recently engineered magnetic nanoparticulate probes targeting clinically promising molecular and cellular players/processes, involved in early atherosclerotic lesion formation to plaque rupture and erosion.

Mimtags: the use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.

Luminescent probes for the bioimaging of small anionic species in vitro and in vivo

The ability to spatiotemporally identify the formation of specific anionic species, or track changes in their concentration inside living systems, is of critical importance in deciphering their exact biological roles and effects. The development of probes (also called bioimaging agents and intracellular sensors) to achieve this goal has become a rapidly growing branch of supramolecular chemistry. In this critical review the challenges specific to the task are identified and for a select range of small anions of environmental and biological relevance (fluoride, chloride, iodide, cyanide, pyrophosphate, bicarbonate, hydrosulphide, peroxynitrite, hypochlorite and hypobromite) a comprehensive overview of the currently available in vitro and in vivo probes is provided.

From "try it and see" to strategic exploration: characterizing young children's scientific reasoning

This article explores ways of characterizing different dimensions and levels of scientific reasoning in early elementary school children, in the context of open explorations. The article focuses on children's performance on three probes which involve using evidence to generate and evaluate knowledge claims. A number of dimensions have been used to characterize children's approaches to exploration and knowledge construction, which demonstrate the interrelationship between conceptual knowledge and scientific reasoning. Children differed markedly across these dimensions, yet individual children were relatively consistent in their approach to the tasks. The major differences in performance are linked to fundamental distinctions in the way ideas are viewed in relation to evidence.

Lupin kernel fiber consumption modifies fecal microbiota in healthy men as determined by rRNA gene flourescent in situ by hybridization

Background Changes in the composition of gastrointestinal microbiota by dietary interventions using pro- and prebiotics provide opportunity for improving health and preventing disease. However, the capacity of lupin kernel fiber (LKFibre), a novel legume-derived food ingredient, to act as a prebiotic and modulate the colonic microbiota in humans needed investigation.

Aim of the study The present study aimed to determine the effect of LKFibre on human intestinal microbiota by quantitative fluorescent in situ hybridization (FISH) analysis.

Design A total of 18 free-living healthy males between the ages of 24 and 64 years consumed a control diet and a LKFibre diet (containing an additional 17–30 g/day fiber beyond that of the control—incorporated into daily food items) for 28 days with a 28-day washout period in a single-blind, randomized, crossover dietary intervention design.
Methods Fecal samples were collected for 3 days towards the end of each diet and microbial populations analyzed by FISH analysis using 16S rRNA gene-based oligonucleotide probes targeting total and predominant microbial populations.

Results Significantly higher levels of Bifidobacterium spp. (P = 0.001) and significantly lower levels of the clostridia group of C. ramosum, C. spiroforme and C. cocleatum (P = 0.039) were observed on the LKFibre diet compared with the control. No significant differences between the LKFibre and the control diet were observed for total bacteria, Lactobacillus spp., the Eubacterium spp., the C. histolyticum/C. lituseburense group and the Bacteroides–Prevotella group.
Conclusions Ingestion of LKFibre stimulated colonic bifidobacteria growth, which suggests that this dietary fiber may be considered as a prebiotic and may beneficially contribute to colon health.

Skin temperature as a noninvasive marker of haemodynamic and perfusion status in adult cardiac surgical patients : an observational study

Objective
Foot temperature has long been advocated as a reliable noninvasive measure of cardiac output despite equivocal evidence. The aim of this pilot study was to investigate the relationship between noninvasively measured skin temperature and the more invasive core-peripheral temperature gradients (CPTGs), against cardiac output, systemic vascular resistance, serum lactate, and base deficit.

Research methodology
The study was of a prospective, observational and correlational design. Seventy-six measurements were recorded on 10 adults postcardiac surgery. Haemodynamic assessments were made via bolus thermodilution. Skin temperature was measured objectively via adhesive probes, and subjectively using a three-point scale.

Setting
The study was conducted within a tertiary level intensive care unit.

Results
Cardiac output was a significant predictor for objectively measured skin temperature and CPTG (p = .001 and p = .004, respectively). Subjective assessment of skin temperature was significantly related to cardiac output, systemic vascular resistance, and serum lactate (p < .001, respectively).

Conclusions
These results support the utilisation of skin temperature as a noninvasive marker of cardiac output and perfusion. The use of CPTG was shown to be unnecessary, given the parallels in results with the less invasive skin temperature parameters. A larger study is however required to validate these findings.

Dresden as Opferstadt? : the politics of commemorating destruction, 1985-2005

Using public remembrance of the controversial WWII firebombing of Dresden as its case study, the thesis probes the politics of war memory and commemoration. It argues that before, during, and after Germany's reunification, Dresden was portrayed as a, if not the, leading paradigm of German wartime loss and suffering.

An investigation into transition metal ion binding properties of silk fibers and particles using radioisotopes

Silk is a structural protein fiber that is stable over a wide pH range making it attractive for use in medical and environmental applications. Variation in amino acid composition has the potential for selective binding for ions under varying conditions. Here we report on the metal ion separation potential of Mulberry and Eri silk fibers and powders over a range of pH. Highly sensitive radiotracer probes, ⁶⁴Cu²⁺, ¹⁰⁹Cd²⁺, and ⁵⁷Co²⁺ were used to study the absorption of their respective stable metal ions Cu²⁺, Cd²⁺, and Co²⁺ into and from the silk sorbents. The total amount of each metal ion absorbed and time taken to reach equilibrium occurred in the following order: Cu²⁺ > Cd²⁺ > Co ²⁺. In all cases the silk powders absorbed metal ions faster than their respective silk fibers. Intensive degumming of the fibers and powders significantly reduced the time to absorb respective metal ions and the time to reach equilibrium was reduced from hours to 5-15 min at pH 8. Once bound, 45-100% of the metal ions were released from the sorbents after exposure to pH 3 buffer for 30 min. The transition metal ion loading capacity for the silk sorbents was considerably higher than that found for commercial ion exchange resins (AG MP-50 and AG 50W-X2) under similar conditions. Interestingly, total Cu²⁺ bound was found to be higher than theoretically predicted values based on known specific Cu²⁺ binding sites (AHGGYSGY), suggesting that additional (new) sites for transition metal ion binding sites are present in silk fibers.