Quantitative analysis of the metabolism in the isolated pupal abdomens of the silkworm, Bombyx mori, infected with nuclear polyhedrosis virus

The rate of oxygen uptake and the amount of several main cell constituents were determined in NPV-infected and uninfected isolated pupal abdomens of the silkworm, Bombyx mori. The oxygen uptake in uninfected isolated abdomens increases for the first 2 days due to the effect of water injection, but thereafter it remains almost unchanged at a relatively low level. When isolated abdomens are infected with an NPV, the oxygen uptake increases markedly from 3 days postinfection onward, being about three times that of uninfected isolated abdomens at late stages of infection. The amounts of cell constituents analyzed in the present study change little in uninfected isolated abdomens throughout the experiment. Infection of NPV leads to a marked accumulation of both DNA and RNA, and a loss of glycogen. The amount of protein, lipid, and sugar are scarcely affected by NPV infection. These results indicate that the activity of metabolism in uninfected isolated abdomens is not only low but also stable, and that infection of NPV results in an activation of host cell metabolism. It is considered that such isolated pupal abdomens provide an excellent opportunity to analyze both the process of NPV replication and the alteration of host cell metabolism resulting from NPV infection.     

Direct studies of dikaryotization in Schizophyllum commune

The growth, duplication and fate of multikaryotic hyphae bearing true clamp connections, as derived from compatible matings of Schizophyllum commune, were studied by phase contrast microscopy. The nuclei (N) of multikaryotic apices maintained a near central position during hyphal growth. True clamp connection formation occurred with near synchronous mitosis followed by septal synthesis across the clamp neck and main hyphal axis. Nuclear progeny after mitosis in a hexakaryon included 6 N in the apex, 1 N in the clamp and 5 N in the penultimate cell; the solitary nucleus in the clamp later entered the penultimate cell. Similar events occurred for clamp connection formation and mitosis in the trikaryon, quadrikaryon or pentakaryon, whether in the apex or primary branches. Nuclear content of the multikaryotic apex (2 N through 10 N) had no apparent effect on the rate of individual hyphal growth. Reduction of the nuclear number in a trikaryon occurred by long-term entrappment of a solitary nucleus in the clamp and subsequent outgrowth of the dikaryotic penultimate cell. Occasionally, more than one nucleus became entrapped in the clamp cell. The ephemeral nature of the multikaryon was indicated by the fact that older cultures appeared to be exclusively dikaryotic hyphae at the colony periphery.     

Detection by electron spin resonance of an exchange-coupled Cob(II)alamin...free radical pair species generated by anaerobic photolysis of polycrystalline adenosylcobalamin

Exposure to nitrous oxide (N₂O) $\text{in vivo}$ is accompanied by oxidation of cob[I]alamin to the inactive cob[III]alamin [1] and to loss of methionine synthetase activity [2]. There is a steady increase in thymidylate synthetase activity in marrow collected from rats exposed to N₂O and this returns to normal on restoring the animals to an air environment.     

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. $\text{249}$, 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. $\text{8}$, 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m₃², 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.     

Azocarcinogen-induced release of chromatin-associated RNA into liver cytoplasm: the possible role of reiterated RNA sequences in the control of RNA processing

In the liver of rats fed the azocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) reiterated RNA sequence transcribed from middle repetitive DNA are released into the cytoplasm. The same repetitive nucleotide sequences can be isolated from the chromatin of the liver of control animals in the form of metabolically highly active, 13 000 daltons RNA. This small, chromatin-associated RNA originates from nuclear RNA larger than 10 S. The discontinuation of the feeding of the azocarcinogen will not stop the release of the nuclear reiterated RNA sequences into the cytoplasm. The repetitive sequences of nuclear RNA which are released into the cytoplasm in animals fed the azocarcinogen can no longer be found in the chromatin in the form of small RNA molecules. The results can be explained by the assumption that the reiterated RNA sequences are involved in the upholding of RNA processing. A cell-specific processing of RNA will be maintained by the interaction of reiterated RNA fragments from already processed RNA with the reiterated complementary sequences on RNA yet to be processed. Existence of such a feed-back circuit would make it possible to explain how a temporary interference of the azocarcinogen with RNA processing will result in the disappearance of specific reiterated RNA sequences from the chromatin. It could also explain the continuation of the release of the same repeated RNA sequences into the cytoplasm as part of larger RNA molecules even after the removal of the carcinogen.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Synthesis and relative stereochemistry of the four mercapturic acids derived from styrene oxide and N-acetylcysteine

The chemical reaction between (±)-styrene oxide and N-acetylcysteine produces both positional isomers ($\text{1}$ and $\text{2}$) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer $\text{1}$ (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (?)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of $\text{1}$ and $\text{2}$ were separated by high pressure liquid chromatography (HPLC) and identified by ¹H- and ¹³C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers $\text{3}$–$\text{6}$ should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide.     

Induction of both cytochromes P-450 and P-448 by 2,3',4,4',5-pentabromobiphenyl, a component of fireMaster

The synthesis and purification of a component of fireMaster BP-6 and fireMaster FF-1, 2,3′,4,4′,5-pentabromobiphenyl, is described. The compound was found to be a potent inducer of liver microsomal drug-metabolizing enzymes in the rat, enhancing those enzymic activities induced by both phenobarbitone and 3-methylcholanthrene (i.e. cytochromes P-450 and P-448). The pentabromobiphenyl enhanced the activities of benzo[a]pyrene hydroxylase, dimethylamino-antipyrine N-demethylase and NADPH-cytochrome c reductase. The hepatic cytochromes b₅ and P-450 were increased and the Soret peak maximum of the latter was shifted to 448.5 nm. The relative peak intensities and spectral shifts for the ethylisocyanide-binding difference spectra confirmed the mixed induction characteristics of 2,3′,4,4′,5-pentabromobiphenyl.     

Polychlorinated biphenyl isomers and congeners as inducers of both 3-methylcholanthrene- and phenobarbitone-type microsomal enzyme activity

Highly purified synthetic polychlorinated biphenyls substituted in the meta and para positions of both phenyl rings and at one ortho position were administered to male Wistar rats and the effects of these compounds on the microsomal drug-metabolising enzymes were evaluated. The in vivo effects of these compounds were determined by measuring the microsomal benzo[a]pyrene hydroxylase, dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC), 2,2',4,4'-tetrachlorobiphenyl (TCBP-II) (a PB-type inducer), 3,3',4,4'-tetrachlorobiphenyl (TCBP-I) (an MC-type inducer), PB plus MC (coadministered) and TCBP-II + TCBP-I (coadministered) to the test animals. At dosage levels of 30 and 150 mumol . kg-1, pretreatment with 2,3,3',4,4'-pentachlorobiphenyl (PCBP-II), 2,3',4,4',5-pentachlorobiphenyl (PCBP-I), 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-II) and 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-III) gave hepatic microsomes with enzymic and spectral properties consistent with a mixed pattern of induction. These polychlorinated biphenyl (PCB) isomers and congeners have been identified as either major or minor components of the commercial PCB mixtures and must contribute to their activity as MC-type inducers. The only PCB isomer in this series which was not a mixed type inducer was 2,3',4,4',5,5'-hexachlorobiphenyl (HCBP-I) which appeared to be a PB-type inducer. This contrasted to the mixed-type activity observed for 2,3',4,4',5,5'-hexabromobiphenyl which was isolated from a commercial polybrominated biphenyl (PBB) mixture.     

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.