Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

Summary The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combintion, of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10⁻⁵ M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. This work was supported in part by Grants CA-07175, CA-22484, and CA-17334 from the National Cancer Institute. D. P. G. is a Predoctoral Fellow at the Food Research Institute, supported by a fellowship from the Monsanto Fund and by NIH Grant R01-AI 15693 to Prof. Michael W. Pariza, Food Research Institute, University of Wisconsin, Madison.     

Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences.

Six purified glycosyltransferase (a beta-galactoside alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 2 leads to 3 sialyltransferase, an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase, a beta-galactoside alpha 1 leads to 2 fucosyltransferase, a beta-N-acetylglucosaminide alpha 1 leads to 3 fucosyltransferase, and a (fucosyl alpha 1 leads to 2) galactoside alpha 1 leads to 3 N-acetyl-galactosaminyltransferase) have been used to study the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences in mammalian glycoproteins. The two glycoproteins used as model acceptor substrates in this study were human asialotransferrin, which contains the nonreducing terminal oligosaccharide sequence Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man, and antifreeze glycoprotein, which contains oligosaccarides with the structure, Gal beta 1 leads to 3GalNAc alph 1 leads O-Thr. Sequential action of the six glycosyltransferases on these model substrates led to the formation of previously described oligosaccharide structures. The studies reported here indicate that the substrate specificities of the individual enzymes dictate the structures that can be synthesized and the pathways by which they may be formed. The actions of a number of the transferasesare mutually exclusive, thereby prohibiting the formation of theoretically possible oligosaccharide structures. Oligosaccharides with the terminal sequence NeuAc alpha 2 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc cannot be formed because the prior incorporation of sialic acid by the sialyltransferases yields products that are not acceptor substrates for the fucosyltransferases, and vice versa. Synthesis of other products requires that the enzymes act sequentially in a specific order. The structures NeuAc alpha 2 leads to 6(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, Fuc alpha 1 leads to 2Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc, GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4GlcNAc, and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3GalNAc can only be synthesized if the fucosyl alpha 1 leads to 2 galactose linkage is formed first. Synthesis of the pentasaccharide sequences GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc and GalNAc alpha 1 leads to 3(Fuc alpha 1 leads to 2)Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc requires that the N-acetylgalactosaminyltransferase act last on the former structure and that the alpha 1 leads to 3 fucosyltransferase act last on the latter. In those instances where a product can be formed by one of two possible pathways, the comparisons of reaction rates indicate that one pathway is usually preferred...     

Responses of the spider Argiope aurantia to low frequency phasic and continuous vibrations

I maintained female orb-weaving spiders, Argiope aurantia, in the laboratory and subjected them to low frequency phasic and continuous transverse vibrations of known frequency, amplitude, and position on the web. Stimuli were administered for periods of 1 min and all behaviour recorded. Pumping was associated with tactile contact to the spider's abdomen. Pumping increased with increasing frequency of phasic stimuli delivered to the web surface. Attack increased linearly with the log of stimulus frequencies between 0 and 14 Hz for stimuli delivered to the catching spiral. Dropping occurred only when high frequencies and amplitudes of stimuli were administered to the spider's abdomen and the hub of the web.     

Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.     

A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.