Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.     

Action of Inhibitors of Ammonia Assimilation on Amino Acid Metabolism in Hordeum vulgare L. (cv Golden Promise)

Barley (Hordeum vulgare L. cv Golden Promise) plants were grown in a continuous culture system in which the root and shoot ammonia and amino acid levels were constant over a 6-hour experimental period. Methionine sulfoximine (MSO), 1 millimolarity when added to the culture medium, caused a total inactivation of root glutamine synthetase with little effect on the shoot enzyme. Root ammonia levels increased and glutamine levels decreased, irrespective of whether the plants were grown in 1 millimolar nitrate or 1 millimolar ammonia. Levels of glutamate, aspartate, serine, threonine, and asparagine all increased. There was little alteration in the amino acid and ammonia levels in the shoot, suggesting that MSO is not rapidly transported.

The addition of azaserine (25 micrograms per milliliter) to nitrate-grown plants caused a rapid increase in root ammonia, glutamine, and serine levels with a corresponding decrease in glutamate, aspartate, and alanine. Glutamine levels also increased in the shoot.

The in vivo effect of MSO and azaserine was as would be predicted by their known in vitro inhibitory action if the glutamine synthetase/glutamate synthase pathway of ammonia assimilation was in operation.

     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human V_H antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin V_H fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIal, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIal and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.     

Characterization of the protein from gas-vacuole membranes of the blue-green alga,Microcystis aeruginosa

Summary The purified protein which constitutes the membranes of the gas vacuoles of the the blue-green alga, Microcystis aeruginosa, was partially characterized. Gel electrophoresis and end-group analysis indicate that the protein is a single species. Strongly protic solvents such as formic acid are the only reagents causing appreciable solubilization of the membrane protein. Infrared spectroscopy shows that the membrane protein has both -helix or random-coil conformation, and -conformation.This work was supported by the U. S. Atomic Energy Commission under Contract AT-(11-1)-1338.     

Evolution of a New Gene Substituting for the leuD Gene of Salmonella typhimurium: Characterization of supQ Mutations

Alpha-isopropylmalate isomerase, the second specific enzyme in the biosynthesis of leucine, is coded for by two genes, leuC and leuD. Leucine auxotrophs, harboring leuD mutations including a deletion of the entire leuD gene, revert to leucine prototrophy owing to mutations at a locus, supQ, substantially distant to the leucine operon. A large number of independently isolated supQ mutations were characterized. A significant increase in the spontaneous frequency of supQ mutations was found after mutagenesis with 2-aminopurine, N-methyl-N′-nitro-N-nitrosoguanidine, diethyl sulfate, and nitrous acid. The supQ function in most of these strains is temperature sensitive, resulting in more efficient suppression with decreasing temperature. At higher temperatures, the supQ limits the growth rate of leuD supQ mutant strains. All supQ mutations are co-transducible with proA and proB, with co-transduction frequencies ranging from 5.4 to 99.9% for different supQ mutations. Many supQ mutations were isolated, especially after nitrous acid mutagenesis, that had acquired a simultaneous proline requirement. The data support the idea of two genes, supQ and newD, whose protein products form a complex. The newD gene product, without any genetic alteration, is capable of substituting for the missing leuD protein. However, mutations in the supQ gene (point mutations or deletions) are necessary to make the newD protein available, which is normally tied up in a complex with the supQ protein.     

Effects of formamidoxime on macromolecular synthesis in regenerating liver and hepatomas

Formamidoxime caused an inhibition of [³H]thymidine incorporation into DNA in regenerating liver and transplanted hepatomas of different growth rates when administered by i.p. injection to rats. A dose level of formamidoxime (500 mg/kg body weight) which caused at least a 75% inhibition of DNA synthesis in these tissues had little or no effect on the incorporation of [³H]orotate into total RNA. After administration of formamidoxime there was no significant effect on amino acid nitrogen concentration in the tissues. The incorporation of ³H-labeled amino acids into acid-soluble material, cytoplasmic proteins and acid-insoluble nuclear proteins were either unaffected or showed only small changes after treatment of rats with the drug. In regenerating rat liver and Morris hepatomas 7787 and 7777, formamidoxime caused an inhibition of incorporation of ³H-labeled amino acids into both lysine-rich and arginine-rich histones. In the host livers of rats bearing the transplanted hepatomas, histone synthesis was less affected. The data indicated that formamidoxime causes inhibitory effects which are similar in nature and extent to those previously shown for the structurally related compound, hydroxyurea, in the regenerating rat liver and demonstrated that these effects can also be observed in liver tumors.     

Untersuchungen zur Inkompatibilitt imCulex pipiens-Komplex

Zusammenfassung Bei Kreuzungen zwischenCulex pipiens-Popalationen verschiedener geographischer Herkunft werden drei Kreuzungstypen festgestellt: normale Kreuzbarkeit, reduzierte Kreuzbarkeit und Inkompatibilitt (Nichtkreuzbarkeit). Die drei Kreuzungstypen sind mit Hilfe der Embryonierungsrate, der Schlüpfrate und der entstehenden Nachkommenschaft gegeneinander abgrenzbar. Bei Inkompatibilitt sind 99,9% der Embryonen letal, und etwa 0,1% der Tiere schlüpfen und sind fertile, diploide Weibchen.Die Aktivierung des Eies und der Entwicklungsansto\ erfolgt durch das Spermium. Das Spermium gelangt nicht zur Karyogamie mit dem Pronucleus. Es liegt induzierte, meiotische Parthenogenese vor.Die diploiden, parthenogenetischen Weibchen gehen aus einer Oocyte 2. Ordnung oder aus Teilungsprodukten einer Oocyte 2. Ordnung hervor. Die letalen Embryonen sind haploid. Das Spermium beteiligt sich nicht am Aufbau des Embryos. Nach Aktivierung des Eies wird das Spermium im Eiplasma resorbiert, whrend der haploide Pronucleus einen rein haploiden Embryo aufbaut.Bei einigen Kreuzungen entwickeln sich bis zu 75% der Embryonen bis zum Stadium der histologischen Differenzierung der Organe. Die Embryonen zeigen noch einige Stunden nach dem normalen Schlüpftermin Muskelkontraktionen, vermögen die Eihülle jedoch nicht zu sprengen.Die DNS-Verteilungen von Interphasekernen der letalen Embryonen liegen zwischen den Werten C und 2C für haploide Zellen. ltere Embryonen besitzen in geringem Ma\e höhere DNS-Werte als 2C. Whrend der histologischen Differenzierung der Organe liegen in diploiden Kontrollembryonen und in letalen Embryonen die Ploidiestufen C, 2C, 4C, 8C and 16C vor. Bei inkompatiblen Kreuzungen wird das in das Ei eindringende Spermium cytophotometrisch nachgewiesen. Die Wanderung des Spermiums im Ei wird untersucht.In Normalkreuzungen wird nur ein geringer Grad von Polyspermie festgestellt. Monound Dispermie sind am hufigsten. Es werden die Möglichkeiten diskutiert die zur Blockierung der Karyogamie in inkompatiblen Kreuzungen führen können.

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Investigations on the incompatibility in theCulex pipiens-complex

Summary In crosses between populations of the mosquitoCulex pipiens of different geographical origin three crossing types have been found (1) crosses with normal offspring (2) crosses with reduced offspring and (3) crosses that show almost total incompatibility. In the case of incompatible crosses 99.9% of the embryos are lethal and only about 0.1% of the embryos hatch and develop to fertile diploid females. Based on genetical and cytological data it is argued that induced meiotic parthenogenesis takes place. The sperm does not play any part in the production of the diploid females and the lethal embryos. After the activation of the egg the sperm moves to the center of the egg but it does not succeed in fusing with the pronucleus. As a result the pronucleus starts to develop into a haploid embryo in about 99.9% and only in a few cases the diploidy is restored by a change in the meiotic process in the egg. Up to 75% of the haploid embryos develop to the stage of histological differentiation. The frequency distribution of the DNA in interphase nuclei of these embryos shows a maximum at C and 2 C characteristic for haploid cells. The absence of ploidy classes higher than 2 C in the early embryos is in agreement with the assumption of pure haploidy. After histological differentiation ploidy classes C to 16 C can be found in tissues that show endomitotic growth. The development of the haploid embryos is described. It has been shown through cytophotometric methods that in incompatible crosses entrance of the sperm into the egg takes place. In normal crosses polyspermy is rather rare, monospermy and dispermy are most common. The blocking of the sperm in incompatible crosses is discussed.

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Teil einer Dissertation der Math.-Nat. Fakultt der Universitt Mainz. Herrn Prof. Dr. H. Laven danke ich für die Bereitstellung des Untersuchungsmaterials sowie für die Anregungen bei der Durchführung der Arbeit.     

An estradiol-dependent protein from chicken liver binds single-stranded DNA and RNA

We have identified two estradiol-dependent single-stranded DNA binding proteins in the nucleus and cytoplasm of chicken hepatocytes that bind the sequence 5'TCACCTTCGCTATG3' in the first exon of the chicken vitellogenin gene. As judged by chromatography on heparin-Sepharose and by proteolytic clipping bandshift assay, the two proteins are different. Furthermore, they only bind to the oligonucleotide corresponding to the upper strand. Depurination and depyrimidination interference experiments with the cytoplasmic protein show that the bases CCTT-G are involved in the protein-DNA interaction. An RNA corresponding to the upper strand of the gene between nucleotide positions -73 and +53 competes for binding to the single-stranded DNA. UV cross-linking experiments performed with bromouridine-substituted single-stranded RNA reveal that an estradiol-dependent hepatocyte cytoplasmic protein with a Mr of 71,000 binds to the mRNA-like single-stranded RNA.