Soybean vegetative lipoxygenases are not vacuolar storage proteins

The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A–E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.

Electron microscopy: More than just a pretty picture

In the 1990s, it might have been thought that, for biological sciences, electron microscopy (EM) was a technique that had had its day. The majority of cellular ultrastructure had been described, and many EM units were closed. However, new developments in instruments, including computer control of microscope columns and stages, the introduction of digital cameras with freely avail able software for image processing and manipulation and improvements in specimen prepara tion techniques have led to a resurgence in the use of EM and wider applicability in the biological sciences. This article aims to show how, even in its routine use, EM can provide further insight into cell biology studies, to show how EM is being used to provide fundamental information about the localization and relationships of molecules within cells through the technique of immuno cytochemistry and to introduce techniques such as EM tomography that are providing unrivalled information about structure–function relationships within cells.

Increased magnitude of relaxation to oestrogen in aorta from oestrogen receptor beta knock-out mice

Micromolar concentrations of the biologically active oestrogen 17beta-oestradiol reduce agonist-induced force in vascular preparations through an unidentified mechanism. The aim of the present study was to investigate the importance of oestrogen receptor beta (ERbeta) for oestrogen-induced vascular relaxation. 17beta-oestradiol was added to aortic rings from ERbeta knock-out (-/-) and wild-type (+/+) mice precontracted with noradrenaline. 17beta-oestradiol caused a concentration-dependent (1-100 microM) relaxation of aortic rings from both -/- and +/+ animals of both sexes. Rings from male and female -/- mice were more sensitive to 17beta-oestradiol than those from +/+ mice. Medial thickness, determined by computerized image analysis, was similar in rings from -/- and +/+ animals. Endothelium, as determined by immuno-cytochemistry, was present in -/- and +/+ aorta. Maximal noradrenaline evoked force and sensitivity to noradrenaline were similar in both groups. In summary ERbeta modulates vascular relaxation to microM concentrations of oestrogen; lack of ERbeta renders the vascular wall supersensitive to 17beta-oestradiol. Lack of ERbeta caused no change in vascular wall morphology suggesting that this ER subtype is not involved in vascular structure development.

Cellular Redistribution Of Taurine In Osmotically Swollen Rat Hippocampus

Taurine is transported from Purkinje cells into perineuronal glial cells during hypoosmotic swelling and is lost first from pyramidal cells during hypoxia/ ischemia, suggesting in situ cellular redistribution. Cultured isolated astrocytes or neurons and slices from rat hippocampus were treated first in 290 mOsm saline (ISO), then in 200 mOsm (HYPO) with or without taurine for 15 and 60 minutes.Cells and slices were then fixed in 80 mM phosphate buffered (pH 7.4) fixative containing 1.25% paraformaldehyde and 2.5% glutaraldehyde followed by immuno-cytochemistry .Immunostaining was done either directly on the cell layers or on 1 micron araldite/eponate sections (Pelco). After incubation with antibody to taurine, GFAP, SNAP-25, or normal serum, immunoproduct was visualized using step-avidin-DAB (DAKO:LSAB+). Measurements of pyramidal cell diameters were made on toluidine blue-stained sections containing cells with prominent nucleoli.The data suggest that taurine is initially lost from pyramidal cells within 15 minutes in HYPO to maintain normal cell volume, while taurine efflux from glia occurs over a 60 minute time peroid.

Ultrastructure and immunocytochemistry of interstitial cells of the deep muscular plexus in the canine small intestine

In the canine small intestine one of the pacemaker regions that generates electrical activity is located in the deep muscular plexus. Within this region special cells, known as interstitial cells (IC-DMP), are distributed. The ultrastructure and immuno-cytochemistry of IC-DMP were studied to clarify their identification and relationship to smooth muscle cells.Segments of the ileum were perfused through mesenteric arteries with a physiological buffer solution (pH5.0) for 3 min followed by a fixative (2.5% glutaraldehyde, 1.25mM Ca Cl2 and 3% sucrose in 0.05M cacodylate buffer; pH 7.4), postfixed in 1% OsO4 and embedded in Epon 812 for transmission electron microscopy (TEM). For immunoelectron microscopy, segments were perfused with a fixative solution (0.25% glutaraldehyde, 4% paraformaldehyde, 0.2% picric acid, 0.1% Triton X-100 and 0.1M phosphate buffer pH7.4), dehydrated and embedded in glycol methacrylate (GMA). Sections were stained with monoclonal antibody B4 to smooth muscle actin (ICN 1:200), anti-myosin light chain antibody (1:25), anti desmin- antibody(1:200), and anti-vimentin antibody (1:500) respectively. Immunoreactivities were detected by IgG gold (10nm, 6nm).