Inhibition of lipopolysaccharide activation of 7OZ/3 cells by anti-lipopolysaccharide antibodies

We have investigated the ability of mAb against LPS to inhibit LPS-induced activation of 7OZ/3 pre-B cells. The fine specificity and relative affinity of these mAb for lipid A and LPS were also determined. We found that antibodies inhibited only the activity of glycolipids which they bound with relatively high affinity. However, two high affinity antibodies binding to non-lipid A epitopes did not block cellular activation. Some, but not all, relatively high affinity antibodies binding to the lipid A region of the LPS molecule inhibited biologic activity. The inhibitory antibodies bound to at least two distinct epitopes within the lipid A region. These data suggest that LPS interacts with 7OZ/3 cells in a highly specific fashion.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida,Polychaeta)

Summary Early development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis. Offprint requests to: A.W.C. Dorresteijn     

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs

Summary The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3-azido-3-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mm) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent K _m and V _max revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest. Offprint requests to: J. Magdalou     

Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons

External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.     

Technique for hand-rearing and reintroducing rejected cotton-top tamarin infants

During a one year period, seven rejected cotton-top tamarin infants were hand-reared. A hand-rearing program was developed which included the use of heated surrogates, a non-human primate infant formula, special nipples, and a systematic reintroduction into a family group. This regimen was quite successful. The hand-rearing process yielded a success rate of 75.0%, while the reintroduction process had a success rate of 80.0% for reintroduction into a family group and 100.0% survival rate.     

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.     

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz--anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a rope-like structure. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the rope-like structure are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.