Microencapsulation of hybridomas by cellulose sulfate-polydimethyldiallylammonium chloride procedure

Two hybridoma cell lines of human origin and two of murine origin were encapsulated using the cellulose sulfate-polydimethyldiallylammonium chloride procedure. All lines could be cultivated in capsules, but the time of growth and product synthesis were limited. The membranes formed were impermeable to albumin and transferrin over a period of 240 minutes. Therefore, the stop of cell proliferation is assumed to be caused by the lack of at least these two proteins. The cell densities inside the capsules reached a maximum of 4.1 × 10⁶/ml (human line) and 3.9 × 10⁷/ml (murine line). The maximum product concentration in the capsules measured by IgG- and IgM-ELISA was 0.17 mg/ml (human line) and 7 mg/ml (murine line). The calculated corresponding retention was 93% (human IgM) and 52% (murine IgG). The characterization of the product showed that the immunoglobulin was not intact and might have been destroyed during harvest. Using the same cell lines the method was compared with the alginate-PLL procedure originally developed by LIM and SUN [1].     

Organisation de type tétracoralliaire des rides septales de Palaeacis (Cnidaria, Carbonifére)

Plusquellec, Y., Lafuste, J. & Webb, G. E. 1990 10 15: Organisation de type tétracoralliaire des rides septales de Palaeacis (Cnidaria, Carbonifére). Lethaia, Vol. 23, pp. 385–397. Oslo. ISSN 0024–1164. Precedemment signalée chez Kerforneidictyum (Pleurodictyforme dévonien), la rare disposition des rides septales suivant un mode tétracoralliaire est décrite chez un Palaeack sp. du Viséen d'Algérie et chez P. cuneiformis subsp. A du Viseen d'Australie. Chez Palaeacis sp. sont mises en evidence une ride septale cardinale forte en position externe opposée à une ride antipode déterminant un plan de symétrie bilatérale; de part et d'autre une zone systématiquement confuse marque l'emplacement des rides alaires. La reconnaissance des périantipodes prête à discussion et ce secteur présente une originalité certaine par rapport à celui des Tétracoralliaires. Chez P. cuneiformis subsp. A, une coupe transversale montre la présence de quatre rides proéminentes qui fusionnent dans I'axe du corallite et I'existence d'une symétrie bilatérale. La position systématique du genre Palaeacis est discutée: son attribution aux Tétracoralliaires est exclue, ancun caractére n'est incompatible avec le statut de Tabulé, la création d'une unité taxon-omique nouvelle regroupant Palaeacis, Trachypsammia et les Pleurodictyformes est envisageable mais actuellement prématurée. ? Palaeacis, Tabulata, Rugosa, rides septales, systématique, relations deparenré, Viséen. The tetracoral pattern of septal ridges described in Kerforneidictyum (Devonian Pleurodictyum-like coral) is scar'ce in the Tabulata, but has been found in Palaeacis sp. from the Visean of Algeria and in P. cuneiformis subsp. A from the Visean of Australia. Palaeacis sp. shows a strong cardinal septal ridge in peripheral position, just opposite the counter ridge. thus creating a plane of bilateral symmetry: an area on both sides. which is always blurred, indicates the position of the alar ridges. counter-lateral ridges are not easily identified and yield differing interpretation. Compared with the Rugosa this area presents distinctive features. In P. cuneiformis subsp. A, a transverse section exhibits four well-developed septal ridges that coalesce in the axis of the corallite with obvious bilateral symmetry. The systematic position of the genus Palaeacis is questioned: Rugosa it is not; it may belong to Tabulata: however, erecting a new higher taxon for Palaeacis, Trachypsammia and other Pleurodictyum-like corals is premature at our present state of knowledge. ? Palaeacis, Tabulata, Rugosa, septal ridges, systematic. affinities, Visean.     

Evidence for a direct role of nascent basal bodies during spindle pole initiation in the green alga Spermatozopsis similis.

Basal body replication in the naked biflagellate green alga Spermatozopsis similis was analyzed using standard electron microscopy and immunogold localization of centrin, an ubiquitous centrosomal protein, and p210, a recently characterized basal apparatus component of S. similis. Fibrous disks representing probasal bodies appear at the proximal end of parental basal bodies at the end of interphase and development proceeds via a ring of nine singlet microtubules. Nascent basal bodies dock early to the plasma membrane but p210, usually present in basal body-membrane-linkers of S. similis, was already present on the cytosolic basal body precursors. In addition to the distal connecting fiber and the nuclear basal body connectors (NBBC) of the parental basal bodies, centrin was present on the fibrous probasal bodies, in a linker between probasal bodies and the basal apparatus, in the connecting fiber between nascent basal bodies and their corresponding parent, and, finally, a fiber linking the nascent basal bodies to the nucleus. This NBBC probably is present only in mitotic cells. During elongation a cartwheel of up to seven layers is formed, protruding from the proximal end of nascent basal bodies. Microtubules develop on the cartwheel indicating that it temporarily functions as a microtubule organizing center (MTOC). These microtubules and probably the cartwheels, touch the nuclear envelope at both sides of a nuclear projection. We propose that spindle assembly is initiated at these attachment sites. During metaphase, the spindle poles were close to thylakoid-free lobes of the chloroplast, and the basal bodies were not in the spindle axis. The role of nascent basal bodies during the initial steps of spindle assembly is discussed.     

Stem cell expression and development of trunk musculature of lesser‐spotted dogfish (Scyliorhinus canicula) reveal differences between sharks and teleosts

This study compares the trunk skeletal muscle anatomy in 870‐ and 2900‐degree‐day‐old lesser‐spotted dogfish larvae (Scyliorhinus canicula) via haematoxylin/eosin staining as well as immunohistochemistry and Western blot analysis. The results showed poorly differentiated muscle formation in the trunk segments in the younger larvae and fully developed skeletal muscle with a division of red and white cells in the older larvae. The stem cell marker PAX7, which is present in all developmental stages of teleost fish, is only expressed in the younger dogfish. The results show the necessity of examining the skeletal muscle development in sharks to understand the evolutional changes from cartilaginous fishes to teleosts.     

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.     

Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells.

The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.     

Specificity of antibodies established from mammals in rainbow trout (Oncorhynchus mykiss)

This study deals with the question as to whether antibodies established for mammals are also specific for rainbow trout. The reason for this examination is the major economic importance of rainbow trout in aquaculture and the growing scientific attention. However, there are few primary antibodies so far defined for this fish species. Therefore, the aim of the current study was to test the ability of 15 commercially available antibodies for rainbow trout in an indirect immunofluoresence assay to analyse tissue sections of organs. Five commercially available primary antibodies were identified, which were directed against proteins of one or two organs/ cell types in rainbow trout.     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d'Ivoire and Cameroon

We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in C?te d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO(2) and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from C?te d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group.