Development of IVM-IVF produced 8-cell bovine embryos in simple,serum-free media after conditioning or Co-culture with buffalo rat liver cells

Development of 8-cell bovine embryos derived from in vitro matured/in vitro fertilized (IVM/IVF) oocytes was evaluated in two simple, serum-free media (CZB and SOM) with buffalo rat liver cells co-culture (BRLC) or after conditioning compared to a commonly used, serum-supplemented complex medium TCM-199. In a 3 x 4 factorial design, 578 eight-cell embryos were randomly assigned to 12 treatment groups. The factors were: first, type of culture medium (M199/FBS, CZBg and SOM), and second, the use of BRLC (as co-culture or to condition media for 24 hr and 48 hr) and unconditioned media. Development to morula was not affected by the type of medium, but co-culture and 48 hr conditioning within media type resulted in better development when compared to the 24-hr conditioned or unconditioned groups. Blastocyst development in SOM (38.9%) was different (P < 0.05) than in CZBg (46.6%) and M199/FBS (48.7%) and was lowest in the unconditioned group (27.8%) followed by 24 hr conditioned (33.3%), 48 hr (56.3%), and co-culture (59.6%). No blastocyst expansion was observed with unconditioned media and 24 hr conditioned SOM. Significant differences (P < 0.05) were found among all treatment groups except the co-culture and 48-hr conditioned groups. Hatching occurred only with co-culture and 48-hr conditioned groups of M199/FBS and CZBg media. These data show that CZB with glucose conditioned by BRLC monolayers for 48 hr can support the development of IVM/IVF produced bovine embryos to blastocyst compared to culture in TCM-199 with serum. © 1994 Wiley-Liss, Inc.     

Comparison of the cellular internalization of antibodies used either as immunotoxins or in ADEPT

The internalization into tumor cells of two antibodies (C242 and 454A12), which make potent immunotoxins when linked to ricin A-chain, and an antibody (A5B7), which does not make a potent immunotoxin but has proven useful in ADEPT, was evaluated. The 454A12 antibody was rapidly taken into the cells, 50% of the antibody being internalized after 2 h. The C242 antibody was internalized more slowly, approx 50% being taken up by the cells in 24 h. With A5B7, less than 10% of the antibody was internalized after 24 h. Internalization of the C242 antibody was accompanied by the appearance of antibody degradation products in the cell medium after 2 h, and this degradation could be inhibited by addition of a metabolic inhibitor that prevented cell internalization. In contrast, minimal degradation of the A5B7 antibody could be detected up to 24 h after binding to the cells. In conclusion, both 454A12 and C242 antibodies, which make potent immunotoxins, were internalized into tumor cels. The A5B7 antibody, which does not make a potent immunotoxin, was not internalized, and this property may be one reason why A5B7 has proved useful for delivery of enzymes in ADEPT.     

Ciliated epithelium in the gut of larval Atlantic halibut, Hippoglossus hippoglossus

The presence of cilia was documented in the hindgut of larval Atlantic halibut ( Hippoglossus hippoglossus ) with the use of electron microscopy. Cilia were shown to be present as late as 8 and 9 day post hatch. These observations were compared to similar observations in more primitive teleosts.     

Introns and higher-order structure in the evolution of serpins

Summary The serpins are a large family of eukaryotic proteins, many but not all of whose members are proteinase inhibitors. Most members of this family show relatively low sequence identity, but crystal structures determined for 6 different serpins are closely similar. The intron positions of 11 serpins, and the intron sizes in 9 of these 11, have been determined. There is considerable diversity in number, position, and size of introns among these serpins, though subsets show clear similarity or identity. Dendrograms derived from comparisons of DNA and amino acid sequences and of intron positions for the 11 serpins differ from each other and from dendrograms previously derived from protein sequences. These dendrograms are difficult to reconcile exclusively with a loss of introns from a large primordial set during the evolution of the serpin family. The tertiary structure of the serpins does support the idea that this protein family arose from an early recombination event which fused the amino and carboxyl domains. The structure of the carboxyl domain also suggests that an insertion subsequent to the fusion event contributed two strands of -sheet, which complemented three -sheet strands of the amino domain, to complete -sheet A, which is the central secondary structure feature of the serpins. Few of the introns lie between regions of secondary or tertiary structure, and it seems more likely that many were acquired subsequent to the early events of serpin evolution and have undergone multiple insertions, deletions, and migrations since, subject to the constraint of the serpin structure.Abbreviations Api -1-proteinase inhibitor (human) - Aci -1-antichymotrypsin (human) - Agt angiotensinogen (rat) - Oah ovalbumin (chicken) - Gyh gene y (chicken) - At3 antithrombin 3 (human) - Pi1 plasminogen activator inhibitor 1—endothelial (human) - Pi2 plasminogen activator inhibitor 2—placental (human) - Cli Cl inhibitor (human) - Apl antiplasmin (human) - Bz4 Z protein (barley).     

Acceptor stem and anticodon RNA hairpin helix interactions with glutamine tRNA synthetase

The class I glutamine (Gln) tRNA synthetase interacts with the anticodon and acceptor stem of glutamine tRNA. RNA hairpin helices were designed to probe acceptor stem and anticodon stem-loop contacts. A seven-base pair RNA microhelix derived from the acceptor stem of tRNA^Gln was aminoacylated by Gln tRNA synthetase. Variants of the glutamine acceptor stem microhelix implicated the discriminator base as a major identity element for glutaminylation of the RNA helix. A second RNA microhelix representing the anticodon stem-loop competitively inhibited tRNA^Gln charging. However, the anticodon stem-loop microhelix did not enhance aminoacylation of the acceptor stem microhelix. Thus, transduction of the anticodon identity signal may require covalent continuity of the tRNA chain to trigger efficient aminoacylation.     

Confocal laser scanning light microscopy of the extra-thecal epithelia of undecalcified scleractinian corals

The extra-thecal epithelia of cryofixed undecalcified, freeze-substituted polyps of the scleractinian corals Galaxea fascicularis and Tubastrea faulkneri and axial and basal polyps of Acropora formosa have been examined, in anhydrously prepared thick slices, by confocal laser scanning light microscopy. The avoidance of chemical fixation and decalcification makes it possible to determine whether previously seen structures are real or artefactual products of swelling, shrinkage and distortion. All of the epithelia of all the corals examined are characterised by well defined intercellular spaces. Mucocytes are present in all cell layers in Galaxea and Tubastrea but are not present in any cell layers in the axial polyp of Acropora although they are abundant in the oral ectoderm of the basal polyps in this coral. Zooxanthellae are absent in Tubastrea, the epithelia of the exert septa of Galaxea and the axial polyp of Acropora. The calicoblastic ectoderm is generally composed of thin squamous cells with large intercellular spaces. At rapidly calcifying regions such as the tips of the exert septa of Galaxea, the calicoblastic cells are elongated with extensive arborisation of the basal regions of the cells. They are separated by large intercellular spaces and contain numerous fluorescent granules. The apical regions of these cells appear to be closely applied to the surface of the skeleton. There is no evidence of a space between the apical region of the calicoblastic cells and the skeleton.     

The decline of tree diversity on newly isolated tropical islands: A test of a null hypothesis and some implications

Summary Six islands, each less than a hectare in area, were isolated in about 1913 from the mainland of central Panamá by the rising waters of Gatun Lake. By 1980, the diversity of trees on all but one of these islands was far lower than on mainland plots of comparable size. A restricted subset of tree species has spread on these islands, notablyProtium panamense, Scheelea zonensis, Oenocarpus panamanus andSwartzia simplex. We constructed a null model to predict how chance would change tree diversity and the similarity of tree species compositions of different islands, assuming that each mature tree has equal chances of dying and/or reproducing, regardless of its species. This model cannot account for the diminished diversity of the changes in vegetation on these islands: some factors must be favoring a particular set of tree species.Two factors, exposure to wind and absence of mammals, seem needed to bring about the vegetation changes observed on these small islands. Their vegetation shows many signs of wind damage and of adaptation to resist wind, reflecting its exposure to dry season winds and storm winds sweeping across the lake from the west. Their most common tree species appear to have spread because mammals rarely visit these small and isolated islands. Seed of these common species are normally much eaten by mammals and do not need burial by mammals to escape insect attack.A thorough grasp of plant—animal interactions is needed to understand the events that have taken place on these islands. Identifying those keystone animals essential for maintaining plant diversity is a necessary element of reserve design and forest management in the tropics.The US government has the right to retain a non-exclusive, royalty-free license in and to any copyright covering this paper.     

Gonadotropin-releasing hormone (GnRH) pathways and reproductive control in elasmobranchs

Synopsis Immunoreactive (ir) gonadotropin-releasing hormone (GnRH) is localized in many neurons of the terminal nerve (TN) and midbrain tegmentum, while few ir-cells are observed in the preoptic area and ventral hypothalamus. The paucity of preoptic ir-cells may relate to an unusual feature of the elasmobranch pituitary, i.e. a lack of portal control of gonadotropin-producing cells. TN and midbrain GnRH-ir neurons may be major sources of GnRH used to modulate or otherwise control both pituitary and brain cells via delivery through the systemic circulation. These ir-nuclei also appear to directly innervate CNS regions (the preoptic area, habenula and clasper control area of the spinal cord) involved in sexual functions. Important regulatory mechanisms, represented by interactions between GnRH pathways and sex-steroid concentrating neurons, are likely to occur in the preoptic area, habenula and midbrain tegmentum.