A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron microscopy (IEM) has remained in a primordial phase preventing researchers to routinely incorporate this technique into their investigations. Here, in addition to simple but detailed protocols to perform conventional electron microscopy (EM) on plastic embedded sections, we present a new IEM procedure adapted from the Tokuyasu method to prepare cryosections from mildly fixed cells. This novel approach allows an excellent cell preservation and the negatively stained membranes create superb contrast that leads to a unique resolution of the yeast morphology. This, plus the optimal preservation of the epitopes, permits combined localization studies with a fine resolution of protein complexes, vesicular carriers and organelles at an ultrastructural level. Importantly, we also show that this cryo-immunogold protocol can be combined with high-pressure freezing and therefore cryofixation can be employed if difficulties are encountered to immobilize a particular structure with chemical fixation. This new IEM technique will be a valuable tool for the large community of scientists using yeast as a model system, in particular for those studying membrane transport and dynamics.     

Direct and indirect immunolabelling of HeLa cells with quantum dots

With excellent optical properties, quantum dots (QDs) have been made as attractive molecular probes for labelling cells in biological research. In this study high‐quality CdSe QDs prepared in a paraffin–oleic acid system were used as fluorescent labels in direct and indirect detection of carcinoembryonic antigen (CEA), a cancer marker expressed on the surface of HeLa cells. The primary antibody (Ab) (rabbit anti‐CEA8) and secondary Ab (goat anti‐rabbit IgG) were covalently linked to carboxyl‐functioned CdSe QDs, and both the QDs–antibody and QDs–IgG probes were successfully used to label HeLa cells. The present study demonstrates the practicability of CdSe QDs as an attractive type of fluorescent labels for biological applications such as protein probes and cell imaging. Copyright © 2008 John Wiley & Sons, Ltd.     

Immunocytochemical detection of glucagon and insulin cells in endocrine pancreas and cyclic disparity of plasma glucose in the turtle Melanochelys trijuga

The present investigation was carried out to know the seasonal variation in plasma glucose,insulin and glucagon cells during the reproductive cycle of untreated Melanochelys trijuga. Pancreatic endocrine cells were immunochemically localized.Insulin-immunoreactive (IR) cells occurred in groups of 3-20 and were in close apposition, while glucagon-IR cells were distributed individually between the exocrine pancreas or formed anastomosing cords where cells were not intimately attached. Whenever both IR cell types were present together forming an islet,insulin-IR cells formed clusters in the centre with glucagon-IR cells being scattered at the periphery. Glucagon-IR cells seemed to be secretory throughout the pancreas during the reproductive cycle,while insulin-IR cells were found to be pulsating in their secretion. Mean size of the islet was 1.306, 0.184 and 2.558 mm in the regenerative, reproductive and regressive periods,respectively. In general,insulin-IR cells measured 5.18 (mu)m and glucagon-IR cells 5.22 (mu)m in their longest axis. Invariably, glucagon-IR cells were more in number than insulin-IR cells. The fasting plasma glucose level was 69.97 mg% during the regenerative period, which increased to 97.96 mg% during the reproductive period,and reached a peak value of 113.52 mg% in the regressive period.     

Biosynthesis of scopoletin and scopolin in cassava roots during post-harvest physiological deterioration: The E-Z-isomerisation stage

Two to three days after harvesting, cassava (Manihot esculenta Crantz) roots suffer from post-harvest physiological deterioration (PPD) when secondary metabolites are accumulated. Amongst these are hydroxycoumarins (e.g. scopoletin and its glucoside scopolin) which play roles in plant defence and have pharmacological activities. Some steps in the biosynthesis of these molecules are still unknown in cassava and in other plants. We exploit the accumulation of these coumarins during PPD to investigate the E-Z-isomerisation step in their biosynthesis. Feeding cubed cassava roots with E-cinnamic-3,2′,3′,4′,5′,6′-d₅ acid gave scopoletin-d₂. However, feeding with E-cinnamic-3,2′,3′,4′,5′,6′-d₆ and E-cinnamic-2,3,2′,3′,4′,5′,6′-d₇ acids, both gave scopoletin-d₃, the latter not affording the expected scopoletin-d₄. We therefore synthesised and fed with E-cinnamic-2-d₁ when unlabelled scopoletin was biosynthesised. Solely the hydrogen (or deuterium) at C2 of cinnamic acid is exchanged in the biosynthesis of hydroxycoumarins. If the mechanism of E-Z-cinnamic acid isomerisation were photochemical, we would not expect to see the loss of deuterium which we observed. Therefore, a possible mechanism is an enzyme catalysed 1,4-Michael addition, followed by σ-bond rotation and hydrogen (or deuterium) elimination to yield the Z-isomer. Feeding the roots under light and dark conditions with E-cinnamic-2,3,2′,3′,4′,5′,6′-d₇ acid gave scopoletin-d₃ with no significant difference in the yields. We conclude that the E-Z-isomerisation stage in the biosynthesis of scopoletin and scopolin, in cassava roots during PPD, is not photochemical, but could be catalysed by an isomerase which is independent of light.     

The differences in carbon dynamics between boreal dwarf shrubs and Scots pine seedlings in a microcosm study

Aims The ground level of boreal pine forests consists of a dense layer of ericaceous shrubs, herbs, grasses, mosses and lichens. The primary productivity of this forest floor vegetation is notable but the role the most common ericoid dwarf shrub plant species,Calluna vulgaris,Vaccinium myrtillusandVaccinium vitis-idaea, play in carbon (C) cycling in these ecosystems is poorly understood. Here, we studied their C dynamics in detail using plants of similar size (age 14–19 months) in a microcosm study.     

Identification of the Adenosine Uptake Sites in Guinea Pig Brain

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.     

Characterization of the Arabidopsis thaliana Arath;CDC25 dual-specificity tyrosine phosphatase

CDC25 enzymes are dual-specificity phosphatases involved in the regulation of the cell cycle. No CDC25 enzymes have been described in higher plant organisms. We report here the characterization of an Arabidopsis thaliana CDC25 enzyme, constituted by a sole catalytic domain and devoid of the N-terminal regulatory region found in the human CDC25. We describe the recombinant expression in Escherichia coli of the Arath;CDC25 and its purification for activity assay and structure determination by NMR. The recombinant enzyme has a tyrosine phosphatase activity towards an artificial substrate, a NMR characterization equally concludes to its correct folding. The secondary structure of the protein was predicted on the basis of the assigned chemical shift of (1)H, (15)N, and (13)C backbone atoms of the protein. The presence of a metal ion in the C-terminus of this new protein points to a zinc finger, and sequence homology indicates that this new structural element might be conserved in related plant homologs.     

Orientation of the 19S regulator relative to the 20S core proteasome: an immunoelectron microscopic study

Specific labelling with monoclonal antibodies reveals that in regulator-proteasome complexes the asymmetric 19S regulator (PA700) binds to one or both terminal alpha-disks of the cylinder-shaped 20S core proteasome in such a way that its reclining front part is positioned in the vicinity of proteasome subunit alpha6. The protruding rear part of the regulator appears to be situated distal to the sites occupied by the subunits alpha2 and alpha3, respectively. When viewed from beta1/beta1' to beta4/beta4' along the polar 2-fold axis of the 20S proteasome core, the rear part of each 19S regulator cap appears to protrude clockwise. Thus, a defined alignment of the 19S regulator with respect to the single polar 2-fold rotational axis of the 20S core proteasome is obtained.     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.