Correlation between Biosynthesis of Chlorogenic Acid and Flowering in Asparagus Seedlings Induced by Carbamate Compounds

The flowering of Asparagus seedlings induced by carbamate compoundswe had developed was triggered when the chemicals were appliedin such a way that they were active during the period of shootdifferentiation, i.e., 4 to 10 days after seeding. The rateof flowering was closely correlated to the decrease in the chlorogenicacid content of the bud primordium caused by the carbamate treatment.Cytokinins stimulated metabolism in the buds and decreased theinhibitory effect of the carbamates on it. The site of actionof the chemicals appears to be somewhere on the metabolic pathwaythat leads to the synthesis of chlorogenic acid. (Received August 2, 1990; Accepted February 14, 1991)     

The fine structure of the bovine Descemet's membrane with special reference to biochemical nature

Summary A freeze-etch replica method combined with biochemical analyses was used to investigate the ultrastructural organization of the bovine Descemet's membrane.The freeze-etch replica observations revealed that the intact Descemet's membranes were composed of stacks of two-dimensionally arranged hexagonal lattices, in which four components were resolved; (1) round densities as nodes, (2) rod-like structures connecting the densities, (3) randomly oriented fine filaments within the lattices, and (4) amorphous materials covering the lattices.When the membranes were treated with sodium dodecyl sulfate (SDS) and mercaptoethanol, only the amorphous materials were solubilized. However, both the amorphous materials and rod-like structures disappeared in SDS-mercaptoethanol-urea-treated membranes. When the membranes were treated with a very low concentration (0.0005%) of collagenase, rod-like structures and round densities remained insoluble. If the concentration was raised to 0.01%, only the round densities persisted.Comparing these data with the amino acid analysis of each fraction, the following conclusions may be drawn: rod-like structures and fine filaments contain collagenous proteins of different solubility, while round densities and amorphous materials are non-collagenous in nature.     

Dark-field light microscopic study of the flexibility of F-actin complexes

The flexibility of F-actin complexed with saturating amounts of myosin subfragments has been measured by the use of a dark-field light microscope and a high-sensitivity television camera. When dilute solutions of F-actin complexes were observed in the microscope, single filaments in flexural thermal motion were visible to the eye. Images of the fluctuating filaments were recorded on videotapes using the high-sensitivity camera, and these records were used for the analysis of fluctuation to calculate flexibility in the framework of statistical mechanics of thermal fluctuation in semi-flexible rods. The analysis was carried out by two different methods. In method A, we selected many filaments (the entire length appeared near focus occasionally in the limited period of 10 to 100 seconds), measured the mean square end-to-end distance 〈R²〉 of each filament during the period and also its contour length L, and calculated a parameter λ representing flexibility by the equation given by Landau & Lifshitz (1958): $\text{〈R}{}^2\text{〉 =}\text{[2λL ? 1 +}\text{exp}\text{(?2λL)]}\text{2λ}{}^2$. Then, we obtained a value for λ = 0.040 ± 0.010 μm^?1 for the acto-heavy meromyosin filament at 24.0 °C ± 1.0 deg. C, and λ = 0.027 ± 0.005 μm^?1 for the acto-tropomyosin-heavy meromyosin filament at the same temperature.In method B, still photographs were taken of the video screen to collect a great number of filaments or parts of filaments which appeared just in focus over their length, and the contour length L of each filament and the angle θ(L) between the tangents at its two ends were measured, on the basis of the assumption that the whole length of each filament was in a plane perpendicular to the direction of view. The data were treated statistically and the results were approximated with 〈cosθ(L)〉 = exp(?λL), which holds for an ensemble of filaments with flexibility λ but in two-dimensional thermal motion (Landau & Lifshitz, 1958). The λ-values obtained by this method for acto-heavy meromyosin and acto-tropomyosin-heavy meromyosin filaments were both in good agreements with those obtained by method A, confirming the reliability of our measurement.F-actin complexed with a saturating amount of myosin subfragment-1 was examined by method B, and its flexibility was shown to be little different from that of acto-heavy meromyosin filaments.     

Ornithine decarboxylase induction and polyamine levels in the kidney of estradiol-treated castrated male rats

Ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK) activities and polyamine concentrations on the kidneys of male castrated rats were studied following sc injection of estradiol. Estradiol caused an 11-fold increase in ODC activity 24 hours after administration. SAMDC activity doubled but TK activity decreased by two-thirds 2 days after estradiol treatment. The concentrations of polyamines, especially putrescine, showed sharp elevations 2 days following estradiol treatment, 1 day after the peak of ODC activity. The increase in ODC activity was suppressed by cycloheximide and by actinomycin D. Estradiol and diethylstilbestrol (DES), but not progesterone increased ODC activity. Estradiol suppressed ODC activities of liver, thymus, adrenal glands, testes and prostate. A specific estradiol-binding protein was demonstrated in the rat kidney. The dissociation constant (Kd) was 1.64 × 10^?10 M and numbers of binding sites were 31 fmoles/mg protein. Correlation between the binding of estradiol to the cytosol protein and elevation of ODC by estradiol was observed.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

Quantitative structure-activity relationship of cytokinin-active adenine and urea derivatives

The substituent effect of N⁶-alkyl and -aralkyl adenines on the promotion of the growth of tobacco callus was analysed quantitatively using physico-chemical substituent parameters and regression analysis. The results indicated an optimum steric condition for activity in terms of the maximum width of the N⁶-substituents from the bond-axis connecting the N⁶-atom with its α carbon atom. The electron withdrawing effect of the N⁶-substituent enhances the activity. The substituent effect on the cytokinin activity of phenyl- and diphenyl- urea derivatives determined by Bruce and Zwar using the tobacco pith-block assay was also analysed. The results suggest that position-specific steric and hydrophobic effects of aromatic substituents participate in the variation in activity rationalizing the general trend of the activity; meta >para >ortho derivatives, for both series of compounds. The electronic effect is significant for the activity of diphenylureas but not for that of phenylureas which show somewhat different modes of interaction between the two series at the site of action. Based on inferences made from the correlations, hypothetical maps for the mode of interaction of these three sets of compounds at the site of action have been proposed.     

Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

Unique hairpin structures occurring at the replication origin of phage G4 DNA

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.     

Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli α-hemolysin membrane translocation system

Summary Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli -haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a -sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system. Correspondence to: H. Nakano     

The relationship between oviposition and spontaneous discharges of motor neurons in Bombyx mori

The experiments in this paper were planned to study the mechanisms of release of ovipositional behaviour in bombyx mori. For this purpose, egg-laying and the spontaneous discharges of motor neurons in G IX were compared between mated and virgin females. The spontaneous discharges of motor neurons in G IX were recorded extracellularly from the nerve bundles of the isolated G IX bathed in a mixture of Ringer's solution and body fluid.The mating stimulated egg-laying so definitively that within 24 hr from the removal of males the mated females laid most of the eggs stored in the genital organs. Ageing of the moth accelerated the egg-laying of the virgins: the number of unfertilized eggs laid by virgins within 2 days from emergence was 10 to 20 per cent of the number of fertilized eggs laid by the mated females within 24 hr, but the number of unfertilized eggs laid within 7 days by the virgins reached 60 to 70 per cent. On the other hand, mating activated the spontaneous discharges of motor neurons in G IX. Moth age had a slight effect on the spontaneous discharges of motor neurons in G IX of the virgins.In recording the spontaneous discharges of motor neurons in G IX, the only changed factor was the body fluid outside the ganglion itself. Therefore, it was postulated that mating changed the humoral condition of the female. This change may stimulate the motor neurons in G IX, and then the egg-laying of mared females may be activated.