Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

Synthesis of glycosyl halides and glycosides via 1-O-sulfonyl derivatives

The reaction of an aldose derivative containing a free anomeric hydroxyl group with trifluoromethanesulfonic anhydride or methanesulfonic anhydride, in the presence of halide ion and s-collidine, furnishes a glycosyl halide; if an alcohol is then introduced, glycoside synthesis is effected in an overall, “one-pot” reaction. Several α-d-glucopyranosides, including disaccharides, have been prepared in high yield by using 2,3,4,6-tetra-O-benzyl-d-glucose as the aldose, and generating the corresponding glycosyl bromide(s) in situ. As a halide-exchange step is incorporated in the reaction sequence, orthoacetate formation was favored in reactions of 2,3,4,6-tetra-O-acetyl-d-glucose, such as occurs with per-O-acetylglycosyl halides. Methanesulfonic anhydride promotes glycosidation or orthoester formation in the absence of halide ion, as well as in its presence, whereas formation of an intermediate glycosyl halide appears to be necessary in order to moderate the more vigorous reactions of the trifluoro derivative. The analogous reaction of methanesulfonyl chloride with an aldose provides a ready route to glycosyl chlorides. Under the conditions employed for these various syntheses, acid-sensitive protecting groups may be used, including cyclic and acyclic acetals and O-trityl substituents.     

Inverse correlation between species lifespan and capacity of cultured fibroblasts to convert benzo(a)pyrene to water-soluble metabolites

Diploid fibroblasts from lung and skin of eight mammalian species were cultured and the capacity of these cell strains to convert benzo(a)pyrene (BP) to water-soluble metabolites was determined. The rate of conversion is dependent upon culture density and varies widely among different species. There is a very good inverse correlation between species lifespan and the capacity of cultured fibroblasts from these species to metabolize BP to water-soluble forms. Since these cell systems have also shown a good inverse correlation between species lifespan and biological activity of 7,12-dimethylbenz(a)anthracene (DMBA), these data suggest that the capacity to metabolize polycyclic hydrocarbon carcinogens may be closely related to lifespan in mammalian species.     

Extinction of differentiated glial functions in somatic cell hybrids is incomplete

Rat glioma cells of clone C₆ were hybridized in vitro with mouse L cells of clone A9 or with freshly isolated mouse macrophages, and the hybrids were assayed for glial cell functions. C₆ cells expressed high levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP; EC 3.1.4.37), β-hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.30), glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8), and inducibility of GPDH by hydrocortisone (HC). A9 cells and macrophages had very low activities of these functions. Hybrids between C₆ and A9 or between C₆ and macrophages had greatly reduced activities of these functions, but the hybrids expressed significantly higher activities than the non-glial parent. This incomplete extinction was not due to fusion of two glioma cells with one L cell or macrophage. The difference in GPDH activity in the hybrids as compared with the non-glial parent was due to incomplete shut-off of GPDH of the glial parent, and not to an increase in GPDH production by the non-glial genome.     

Differential effects of pentobarbital and ethanol in mice

The duration of the loss of righting reflex (RR) after ethanol, 4 g/kg, intraperitoneally (i.p.), was significantly longer in “long-sleep” (LS) than in “short-sleep” (SS) mice. This effect was shown to be correlated with differences in brain sensitivities to ethanol. In contrast, pentobarbital sodium (PB), 50 mg/kg, i.p., produced a significantly longer loss of RR in SS than in LS mice. The PB concentrations in the brain were the same in both mouse strains at the time of RR recovery suggesting equal sensitivities of the central nervous systems to PB. The rates of disappearance of PB from the blood were the same in both strains, but the apparent volume of distribution of PB in the LS strain was greater than in SS mice.In addition, C57BL/6J mice were found to be more sensitive than DBA/2J mice to PB, 50 mg/kg. In contrast, C57BL mice are known to be less sensitive than the DBA strain to ethanol. The PB concentration in the brain of DBA mice at the recovery of the RR was significantly greater than in C57BL mice. The apparent volumes of distribution of PB were not different in the two strains, but the rate of disappearance of PB from the blood of C57BL mice was significantly greater than for the DBA strain. In conclusion, factors which govern the brain sensitivities of selected mouse strains to ethanol and pentobarbital may not be equivalent.     

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.     

Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast

The red-pigmented fermenting yeast Phaffia rhodozyma contains astaxanthin as the principal carotenoid pigment. Echinenone, 3-hydroxyechinenone and phoenicoxanthin were also isolated and identified; isocryptoxanthin and canthaxanthin were absent. Evidence is presented for a new carotenoid, 3-hydroxy-3′4′-didehydro-β,ψ-caroten-4-one. A possible biosynthetic scheme for the formation of astaxanthin in P. rhodozyma is suggested.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.