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.

Reduction capability of soil humic substances from the Rio Negro basin, Brazil, towards Hg(II) studied by a multimethod approach and principal component analysis (PCA)

This paper characterizes humic substances (HS) extracted from soil samples collected in the Rio Negro basin in the state of Amazonas, Brazil, particularly investigating their reduction capabilities towards Hg(II) in order to elucidate potential mercury cycling/volatilization in this environment. For this reason, a multimethod approach was used, consisting of both instrumental methods (elemental analysis, EPR, solid-state NMR, FIA combined with cold-vapor AAS of Hg(0)) and statistical methods such as principal component analysis (PCA) and a central composite factorial planning method. The HS under study were divided into groups, complexing and reducing ones, owing to different distribution of their functionalities. The main functionalities (cor)related with reduction of Hg(II) were phenolic, carboxylic and amide groups, while the groups related with complexation of Hg(II) were ethers, hydroxyls, aldehydes and ketones. The HS extracted from floodable regions of the Rio Negro basin presented a greater capacity to retain (to complex, to adsorb physically and/or chemically) Hg(II), while nonfloodable regions showed a greater capacity to reduce Hg(II), indicating that HS extracted from different types of regions contribute in different ways to the biogeochemical mercury cycle in the basin of the mid-Rio Negro, AM, Brazil. (c) 2007 Published by Elsevier B.V.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Effect of refrigeration systems upon frozen bull sperm viability assessed by computer-assisted sperm analysis and fluorescent probes

Sperm cryopreservation success depends upon the maintenance of spermatozoa fertility potential. Sperm cells must preserve both integrity and functionality of several cell structures. The stabilization phase must allow the exit of water from the sperm cells via osmosis. This study aimed to compare the effect of refrigeration in the commercial refrigerator (CR) and the transport/refrigeration box (TRB) upon the viability of frozen bull sperm diluted in three different extenders (A, B and C). Ten Nellore bulls, Bos taurus indicus maintained in Artificial Insemination Center were used and the spermatozoa samples was assessed for Plasma Membrane Integrity and CASA evaluation. The stabilization phase (5 degrees C/4 hours) was performed in the CR as well as in the TRB, and then samples were exposed to nitrogen vapor during 20 minutes and then plunged into nitrogen. The statistical analysis was done using the variance analysis and the significance level was set at 5%. In the CR the post-thawing parameters for PM and ALH were higher (p < 0.05) in the extender A (glicine egg-yolk) and extender B (glicine egg-free) when compared with extender C (TRIS egg-yolk). As for BCF, STR and LIN, the parameters were higher (p < 0.05) in extender B than in C. Samples that were stabilized in the TRB presented higher post-thawing parameters (p < 0.05) for PM and LIN in extender A and extender B when compared with C. BCF and STR parameters were higher (p < 0.05) in extemder B when compared with C. Extender B samples had higher (p < 0.05) PMI when stabilized in CR. The findings in this experiment enable us to say that both CR and TRB were effective in keeping the viability of post-thawing bull semen.

Origin and molecular organization of supernumerary chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae) obtained by DNA probes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A rapid technique for analysis of formalin-fixed, paraffin-embedded tissues by fluorescent in situ hybridization with alpha-satellite probes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors. (C) 2011 Elsevier B.V. All rights reserved.

Alternative paths for insertion of probes into high inclination lunar orbits

The dynamics of the restricted three-body Earth-Moon-particle problem predicts the existence of direct periodic orbits around the Lagrangian equilibrium point L1. From these orbits, we derive a set of trajectories that form links between the Earth and the Moon and are capable of performing transfers between terrestrial and lunar orbits, in addition to defining an escape route from the Earth-Moon system. When we consider a more complex and realistic dynamical system - the four-body Sun-Earth-Moon-particle (probe) problem - the trajectories have an expressive gain of inclination when they penetrate in the lunar influence sphere, thus allowing the insertion of probes into low-altitude lunar orbits with high inclinations, including polar orbits. In this study, we present these links and investigate some possibilities for performing an Earth-Moon transfer based on these trajectories. (C) 2007 COSPAR. Published by Elsevier Ltd. All rights reserved.