Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

Summary The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody.The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries.Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile. The authors express their gratitude to Mrs. Elizabeth Santibáñez and Mr. Julio Lamilla for providing the lamprey larvae and to Mr. Humberto Molina for preparing the three-dimensional drawing     

Alpha-DNA. IV: Alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to intercalating oxazolopyridocarbazole. Synthesis, physicochemical properties and poly (rA) binding

A new set of molecules made of an intercalating agent (oxazolopyridocarbazole, OPC) covalently linked through a polymethylene chain of various length to the 3' end of alpha-anomeric or beta-anomeric tetradeoxynucleotides (alpha- or beta-T4) have been synthesized. The beta-thymidylate modified compound (beta-T4C5OPC) is able to interact with the complementary sequence, beta-poly (rA); this interaction is strongly stabilized compared to the parent compound, beta-oligo(dT)4 and is specific for poly (rA). The molecule synthesized from the unnatural alpha-anomer, alpha-T4C5OPC, is also able to interact with poly (rA) leading to the formation of an alpha-beta hybrid stabilized by the energy provided by the OPC moiety. The stoechiometry of the binding reaction shows that an A-T pairing occurs in the alpha-beta heterohybrids. Tm studies reveal that the alpha-beta heterohybrids are more stable than their beta-beta counterparts.     

alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition.

alpha and beta-anomeric d(G2T12G2) oligodeoxyribonucleotides were compared for their hybridization to rA12: the observed melting temperatures are 27 degrees C for beta-oligodeoxyribonucleotide/RNA hybrid and 53 degrees C for alpha-oligodeoxyribonucleotide/RNA. alpha-oligonucleotides with the four bases, complementary to natural mRNAs, were synthesized for the first time, labeled at their 5'-end with [32P] and used as probes in Northern blot experiments. In spite of these higher affinities for their target RNA's, they were unable to block translation of natural or synthetic mRNA's in rabbit reticulocyte lysate. We have studied the RNase H activity on model rA12:alpha- or beta-d(G2T12G2) hybrids or on mRNA:alpha- or beta-oligonucleotides hybrids. Specific hybridization protects RNA strech when using alpha-oligonucleotides but not beta-oligonucleotides. Thus, our results show the inability of RNase H to degrade RNA in alpha-oligodeoxyribonucleotides:RNA duplexes.     

Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

A new mutant class of soybean lacks urease in leaves but not in leaf-derived callus or in roots

Summary We reported earlier the recovery of two classes of soybean urease mutants in soybean (Glycine max L. Merr. cv. Williams). Class I mutants lack the embryo-specific urease while class II mutants lack the activities of both urease isozymes, the embryo-specific and the ubiquitous urease, the latter found in all tissues examined. We report here the recovery of a true-breeding mutant, aj3, which represents the third phenotypic class: normal levels of embryo-specific urease and little or no ubiquitous urease. Unlike class II mutant plants which lack urease in all tissue, aj3 lacks urease activity only in leaves (ca. 2% normal activity); its roots have near normal urease activity. Callus derived from leaves of aj3 has 14% to 40% the urease activity of Williams 82 callus. This partial reduction in urease activity in aj3 callus is sufficient to reduce growth with urea as sole nitrogen source and to confer resistance to 50 mM urea added to callus maintenance medium. Leaves of aj3 produce more than 40 times the urease antigen expected from their urease activity. The aj3 trait is due to a single recessive lesion which is not allelic with lesions at theEu2, Eu3 (class II) orEu1 (class I) loci. We designate the aj3 genotype aseu4/eu4.     

Alpha-DNA VIII: thermodynamic parameters of complexes formed between the oligo-alpha-deoxynucleotides: alpha-d(GGAAGG) and alpha-d(CCTTCC) and their complementary oligo-beta-deoxynucleotides: beta-d(CCTTCC) and beta-d(GGAAGG) are different.

The temperature dependence of the formation of a complex between an alpha-d(CCTTCC) hexanucleotide and its complementary beta-d(GGAAGG) sequence was studied and compared to the formation of the beta-d(CCTTCC):beta-d(GGAAGG) complex. Such alpha-beta complex is more stable than the regular beta:beta complex. The Tm value for the alpha:beta complex is 28 degrees C (delta G degrees = -7.3 kcal/mole) while Tm = 20, 1 degree C (delta G degrees = -6.3 kcal/mole) for the beta:beta complex. The stoechiometry of the alpha:beta complex corresponds to the formation of a 1:1 duplex. However, when the alpha- strand is made of alpha-purines: alpha-d(GGAAGG), the stability of the alpha:beta complex, alpha-d(GGAAGG):beta-d(CCTTCC) is found to be lower (Tm = 13.8 degrees C) than the stability of the regular beta-beta complex, leading to the conclusion that the nature of the alpha-sequence is important in terms of stability when considering the synthesis of such a sequence for using it as antisense oligonucleotide.     

Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster

The genomic distribution of elements of the copia, 412, B 104, mdg 1, mdg 4 and 1731 transposon families was compared by the Southern technique in DNA preparations extracted from brains, salivary glands and adult flies of two related Drosophila lines. The copia, 412 and mdg 1 sequences were also probed in DNA from sperm, embryos, and 1st and 2nd instar larvae. The homogeneity of the patterns observed shows that somatic transposition is unlikely to occur frequently. A correlation between mobility and the euchromatic or heterochromatic location of transposable elements is discussed. In addition, an explanation of the variable band intensities of transposable elements in Southern autoradiographs is proposed.     

The phylogenic position of Peptococcus niger based on 16S rRNA sequence studies

A 1330 base-pair fragment of a 16S rRNA gene has been amplified, cloned and sequenced. Comparison to other 16S rRNA sequences of eubacteria showed that P. niger represents a deep branch within the subdivision "Gram-positive with Gram-negative cell walls". It is not related to peptostreptococci, representatives of this genus studied so far are more closely related to clostridia.     

Cultivation of the sapropelic ciliate Plagiopyla nasuta Stein and isolation of the endosymbiont Methanobacterium formicicum

The sapropelic ciliate Plagiopyla nasuta was isolated and cultured in monoculture. Optimal conditions for growth were: 15–20°C, pH about 7, and about 2% of oxygen in the headspace. Cultures of P. nasuta produced methane. Epifluorescence microscopy revealed the presence of methanogenic bacteria as endosymbionts. An endosymbiont of the ciliate was isolated and identified as Methanobacterium formicicum. In the ciliate cell these methanogens were found to be closely associated with microbody-like organelles. No mitochondria could be detected.