Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.     

Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.     

A comparative 1H NMR study of mouse alpha(1-53) and beta(2-53) epidermal growth factors

The three-dimensional structure of the mouse epidermal growth factor (EGF) in solution was studied by comparison of the 1H NMR spectra of alpha EGF (1-53) and beta EGF (2-53, des-asparaginyl 1 form). Using pH dependence of chemical shifts and a two-dimensional difference spectrum, the effect of the N-terminal deletion was investigated based on the complete assignment of the proton resonances. The affected residues were all found to be located exactly in the triple-stranded, beta-sheet core in the N-terminal domain of the EGF molecule.     

Combination therapy of radiation and Sizofiran (SPG) on the tumor growth and metastasis on squamous-cell carcinoma NR-S1 in syngeneic C3H/He mice

The efficacy of Sizofiran(SPG), a highly purified -1,3-D-glucan from the culture broth of a basidiomycetesSchizophyllum commune Fries, in combination with local irradiation was investigated using squamous-cell carcinoma NR-S1 and syngeneic hosts of OH/He mice. NR-S1 tumor was implanted sc in the thigh of C3H/He mice. When tumor grew to 4 mm in diameter, the local irradiation of 55 Gy was delivered. SPG was injected im at a dose of 5 mg/kg. When SPG was administered after irradiation, remarkable inhibition of tumor growth was observed in comparison with the radiation alone group. Furthermore, the combination effect of radiation and active immunotherapy using mitomycin C-treated NR-S1 cells as vaccine was examined. When radiotherapy and active immunotherapy were combined with SPG, suppression of tumor growth was observed from an early stage in comparison with the group which was not administered SPG. SPG also inhibited the pulmonary metastasis of NR-S1 tumor after radiotherapy.     

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

An anti-carbohydrate monoclonal antibody inhibits cell-substratum adhesion of F9 embryonal carcinoma cells

A monoclonal rat IgM antibody (4C9) raised against F9 embryonal carcinoma cells reacted with fucosyl residues in poly-N-acetyllactosamine-type large carbohydrates of these cells (embryoglycan). The chemical properties and distribution of the antigen resembled those of SSEA-1. The monoclonal antibody was found to inhibit cell-substratum adhesion of F9 cells: in the presence of the antibody, cells grew as spherical cell aggregates on plastic dishes. When the antibody was added to the already spread cells, they displayed the initial sign of rounding up within 3 h; the rounding process was largely completed within 6 h. After removal of the antibody, cells resumed their normal morphology. The antibody could act in the presence of 2,4-dinitrophenol. In serum-free medium, F9 cells spread on plastic dishes coated with fibronectin or with laminin, and the process was also inhibited by the antibody. Immuno-electronmicroscopy revealed that 4C9 antigen was diffusely distributed over the cell surface of F9 cells. The distribution of the antigen was not altered generally after culturing with the antibody for 6 h. Another monoclonal rat IgM antibody, which did not react with embryoglycan and resembled anti-Forssman, did not inhibit cell-substratum adhesion of F9 cells, in spite of its reactivity to the cells. Thus, a glycoprotein with fucosyl (poly)-N-acetyllactosamine structure appears to be involved in cell-substratum adhesion of F9 cells.     

Synergistic defense system by cooperative natural effectors against metastasis of B16 melanoma cells in H-2-associated control: different behavior of H-2+ and H-2- cells in metastatic processes

H-2+ and H-2- cells of B16 melanoma were established by repeated fluorescence-activated cell sorting. The H-2- line formed no metastasis in untreated C57BL/6 mice, whereas the H-2+ cells showed evidence of metastatic development. This difference was ascribed mainly to the increased susceptibility of H-2- cells to attack by natural effector mechanisms, particularly asialo GM1+ NK cells. After treatment with both anti-asialo GM1 serum and whole body irradiation (400 rad), numerous colonies of H-2- cells formed in the lung, whereas the metastasis was only marginally enhanced by irradiation and moderately by treatment with anti-asialo GM1 serum. With the H-2+ cells, treatment with each modality significantly increased the number of metastatic colonies. Therefore collaboration of asialo GM1+ NK cells and radiosensitive natural effectors seems to be the main mechanism involved in the synergistic effects on defense against H-2- cell metastasis, and to a lesser extent against H-2+ cell metastasis. Irradiation (1000 rad) to the right lung to abrogate the organ-associated defense increased the colonies, particularly in the H-2+ cells. On the other hand, treatment with anti-asialo GM1 serum increased colonization in the early phase of metastasis with H-2- cells and may have abolished asialo GM1+ NK cells capable of recognizing the reduced expression of H-2 antigens and eliminating H-2- cells in the blood-born phase. Natural defense mechanisms probably exert suppressive effects on the metastasis of H-2+ cells, mainly in the organ-associated phase after extravasation.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.