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.     

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.     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.     

Macrophage Activation by Tetrahymena pyriformis. I. Active Subcellular Fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H₂O₂) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H₂O₂ release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

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.     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.