Molecular Cloning and Restriction Analysis of EcoRI-fragments of Vicia faba rDNA

EcoRI-fragments of Vicia faba rDNA were cloned in plasmid pBR325.Southern blot hybridization of BamHI-digests of these clonedplasmids and Vicia genomic DNA led to the determination of relativepositions of BamHl sites in the rDNA and the physical map thathad been tentatively made is corrected. (Received May 20, 1982; Accepted July 13, 1983)     

Chronotoxicity of Sodium Valproate and Its Mechanisms in Mice: Dose-Concentration-Response Relationship

A significant circadian rhythm of acute toxicity was demonstrated in mice with intraperitoneal (i.p.) injection of sodium valproate (VPA). The role of pharmacokinetics on the rhythms of the toxicity and electroshock seizure (ES) threshold was investigated. ICR male mice, housed under a light-dark (12 :12) cycle, were injected intraperitoneally 1200 mg/kg for the acute toxicity study and 300 mg/kg for the anticonvulsant effect study. In the acute toxicity, the highest mortality was found when VPA was injected at 1700 and the lowest at 0900 or 0100. The time course of mean plasma and brain VPA concentrations after an injection of VPA was not different between mice injected at 1700 and mice injected at 0100. In the anticonvulsant effect, no significant circadian rhythm was demonstrated for both the ES threshold and the plasma VPA concentrations after i.p. Injection, although a significant rhythm has been reported for them after oral administration. The results suggest that the circadian rhythm in the mortality after an i.p. Injection of VPA may be due to the rhythm in the sensitivity of the central nervous system to the drug and that the mechanism underlying the rhythm of VPA acute toxicity is different from that of the anticonvulsant action of VPA. The route and the time of drug administration are essentially important to study the anticonvulsant effect and acute toxicity of VPA in mice.     

The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min^–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10⁶ cells ml^–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.     

Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Sporocarp ontogeny in Panus (Basidiomycotina): evolution and classification

Ontogenies of cultured Panus conchatus, P. rudis, and P. fulvus sporocarps were observed macroscopically and with scanning electron microscopy. Hymenophore differentiation in Panus involves periclinal growth of context hyphae below a closed surface palisade of hymenial elements, resulting in a cantharelloid appearance and radiate trama. This pattern is qualitatively different from that in Lentinus s. str., which suggests that lamellae of Panus and Lentinus are not homologous. Panus conchatus and P. rudis sporocarps have short stipes, develop directly from the mycelium, and mature in 5–10 d. Panus fulvus sporocarps have an elongate stipe, develop from a pseudosclerotium, and mature in about 3 wk, the first approximately 15 d of which involve apical elongation of a stipelike primordium that is able to dedifferentiate and regenerate cut apices. Panus conchatus and P. rudis sporocarps lacked regeneration ability. Panus conchatus sporocarps developed an ephemeral partial veil that was obliterated during sporocarp expansion. Outgroup comparison suggests that evolutionary changes in developmental programs in Panus have included: 1) delay in offset of primordium growth, with a corresponding increase in primordium size and time to maturation (hypermorphosis); 2) insertion of the pseudosclerotial stage in ontogeny; 3) gain of ability for dedifferentiation and regeneration; and 4) nonterminal gain or loss of veil tissue.