A modified FCR test to evaluate pollen viability in Juniperus communis L.

Pollen viability of Juniperus communis L. and other gymnosperms with taxoid type pollen cannot be assessed with the more common viability tests because the thick sporoderm prevents reagents from penetrating into the cytoplasm. Here we describe a technique for pre‐hydration of pollen that overcomes this problem so that the common FCR test can be used to assess its viability. Pollen of J. communis must be re‐hydrated by suspending in water. This re‐hydration causes the splitting of the exine and a huge swelling of the intine. The sporoderm becomes permeable to fluoresceine diacetate and the FCR‐viability test can be applied. The FCR result is supported by scoring the germination percentage in vitro.     

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.     

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 ± 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 ± 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP₃R₃)_, α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP₃R₃, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP₃R₃, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ～3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10–12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.     

The Liver of Myxine glutinosa: A true tubular gland

Summary The authors have reexamined the liver of Myxine with the light and electron microscopes. The observations demonstrate that the liver in this animal is really a tubular gland, in accordance with the conclusions of older anatomists, but in contrast to more recent statements. The existence of a tubular pattern in the liver of the lowest living vertebrate is important for the elaboration of a valid general model of liver structure, which necessarily has to be based on comparative anatomy.The special cytology of the liver and the ductular cells is presented.     

Synthesis and biological investigation of S-aryl-S-DABO derivatives as HIV-1 inhibitors

S-Aryl-S-DABO derivatives, a novel subclass of S-DABO anti-HIV-1 agents, were synthesized via Ullmann type reaction starting from the corresponding 2-thiouracils by the aid of microwave irradiation. The results of their evaluation as inhibitors of RT are reported together with their antiviral activity in cellular assays.     

HELICAL FILAMENTS IN ASTROCYTIC MITOCHONDRIA OF THE CORPUS STRIATUM IN THE RAT

Astrocytic mitochondria have been studied in serial electron micrographs of the corpus striatum. Special filaments have been found inside several of these mitochondria located in both the cell body and the processes. They were particularly frequently observed in the pericapillary end-feet. The filaments occur within dilated intracristal spaces provided with several communications with the outer chamber, and form helices which are oriented approximately in the same direction. Each filament is about 30 A thick, the diameter of the helix is 140 A, and the pitch is 120 A. Their possible nature and significance are briefly discussed. There is no clear relationship between these mitochondria and other unusual forms of mitochondria previously described in astrocytes from other regions.     

Allosteric Modulation of [3H]CGP 39653 Binding by Glycine in Rat Brain

Abstract: D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pen-tenoic acid (CGP 39653). a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a K_D of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [³H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(±)-2-carboxypiperazin-4-yI]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [³H]CPP binding but inhibits [³H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [³H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [³H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [³H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [³H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.