Effects of combined centrifugation and ultraviolet irradiation of eggs on pattern formation in Chironomus embryos

Summary

Combined mild centrifugation and uv irradiation of Chironomus embryos modified the developmental types expected from centrifugation alone, somewhat differently from the combined strong centrifugation and uv irradiation of Smittia embryos. The modifications changed with the stages irradiated. The change caused by anterior irradiation may depend on whether or not a part of the cytoplasmic zone is irradiated simultaneously with the anterior yolky end; because most of the cytoplasm lies in the posterior half of egg at early irradiation, while the tip of the cytoplasm redistributes near the anterior end by the late irradiation. Early uv irradiation of the anterior end of centrifuged eggs, causing the formation of a double abdomen (DA) or an inverted embryo, is not photoreversible, while the uv damage to the anterior end of uncentrifuged eggs, inducing DA, is. These facts suggest that there is another photoirreversible uv target in addition to the photoreversible target for DA induction or the anterior determinant shown in Smittia. Other changes, such as the induction of a double cephalon by late irradiation of the centrifuged egg, are photoreversible, but in an unusual way in that the level of photorecovery is similar to the result of incubation in the dark after early irradiation, and not to that of the centrifuged controls. These modified results were then compared with those for Smittia embryos.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

Demethylthiolating Agents for Ribonucleoside 5′-S-Methyl Phosphorothiolates

Several oxidizing agents were examined for their ability to demethylthiolate adenosine- and cytidine 5′-S-methyl phosphorothiolates.

Iodine dissolved in an aqueous potassium iodide solution or in dimethyl sulfoxide (DMSO) was the most effective demethylthiolating agent of those tested in the present study, rapidly giving the demethylthiolated products in quantitative yields. The iodine-DMSO solution demethyl-thiolated the ribonucleoside 5′-S-methyl phosphorothiolates to give ribonucleoside 5′-monophosphates even under anhydrous conditions, DMSO acting as an oxygen donor in this reaction.

Hydrogen peroxide has high demethylthiolating ability in spite of its low reaction rate. Isoamyl nitrite, an effective demethylthiolating agent for O-alkyl S-methyl phosphorothiolates, was not effective for the demethylthiolation of ribonucleoside 5′-S-methyl phosphorothiolates, because the unprotected amino groups of the S-methyl nucleotides were attacked by the reagent to give deaminated products. N-Chlorosuccinimide had no effect on the demethylthiolation of S-methyl phosphorothiolates.     

Lipids of Streptomyces sioyaensis

Lipids were extracted with chloroform-methanol from Streptomyces sioyaensis and fractionated on a silicic acid column. Lipids of Streptomyces sioyaensis were mainly composed of neutral lipids, cardiolipin, phosphatidylethanolamines, phosphatidylinositolmonomanno- side and a new lysine-containing lipid.     

Bitter Taste of Synthetic C-Terminal Tetradecapeptide of Bovine β-Casein,H-Pro196-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val209-OH,and Its Related Peptides

The primary structure of bovine β-casein contains the partial sequence of -Pro¹⁹⁶-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val²⁰⁹ in the C-terminal portion. We previously reported that the synthetic C-terminal octapeptide, Arg²⁰²-Val²⁰⁹, is extremely bitter with its threshold value 0.004 mm, 250 times as strong as that of caffeine. To further investigate the bitter taste of the C-terminal portion of β-casein, we synthesized the C-terminal tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, and some of its fragments. A hydrophobic hexapeptide, Pro¹⁹⁶-Val²⁰¹, was twice as bitter as caffeine. The bitter taste of the decapeptide, Pro²⁰⁰-Val²⁰⁹, was the same as that of Arg²⁰²-Val²⁰⁹. Although the tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, was composed of two bitter peptides, Pro¹⁹⁶-Val²⁰¹ and Arg²⁰²-Val²⁰⁹, its bitter taste was weaker than that of Arg²⁰²-Val²⁰⁹ and its threshold value was 0.015 mm. We suggested that the increase of bitterness in peptides through the introduction of hydrophobic amino acids depended on the number of hydrophobic amino acids added. In addition, the synthetic retro analog of Arg²⁰²-Val²⁰⁹ (H-Val-Ile-Ile-Pro-Phe-Pro-Gly-Arg-OH) was not as bitter as Arg²⁰²-Val²⁰⁹. This indicated that the sequence of Arg²⁰²-Val²⁰⁹ is important for extreme bitterness.     

Studies on Nicotinoids as Insecticides

Synthesis of two nicotine isomers, 5′-methylnornicotine and 6-methylnornicotine is described. The latter shows slightly insecticidal action. Myosmine type bases are easily reduced to nornicotine type with sodium borohydride.     

Bitter Taste of H-Pro-Phe-Pro-Gly-Pro-Ile-Pro-OH Corresponding to the Partial Sequence (Positions 61 ~ 67) of Bovine β-Casein,and Related Peptides

The bitter components of cheese are hydrophobic peptides which are produced during the process of enzymatic digestion, and some of the isolated bitter peptides are derived from the middle portion of β-casein. However, quantitative examination of the bitter taste is seldom performed. We synthesized two hydrophobic peptides, H-Pro⁶¹-Phe-Pro-Gly-Pro-Ile-Pro⁶⁷-OH and H-Tyr⁶⁰-Pro-Phe-Pro-Gly-Pro-Ile⁶⁶-OH, which correspond to common portions among the isolated bitter peptides, in order to determine how bitter they were. From the results of sensory analysis, it was found that the synthetic peptides exhibited a bitter taste with threshold values 0.25 and 0.16mm, respectively. We also synthesized their fragments and analogs, and discussed the structure-bitterness relationship.     

Subunits and Sulfhydryl Groups of Beef Liver d-Glycerate Dehydrogenase

The amino acid composition of beef liver d-glycerate dehydrogenase (EC 1.1.1.29) was determined. Results of sodium dodecyl sulfate gel electrophoresis and measurements of the number of NADH bound by the enzyme and the number of the essential sulfhydryl groups suggested that the enzyme was composed of two identical subunits with the molecular weight of 36,000. Close relation between the essential sulfhydryl groups and the coenzyme binding site was also suggested. Effect of an alkylating agent (bromopyruvate) with the structure similar to the substrate was studied. Effects of iodoacetate and iodoacetamide were also studied. It was suggested that these reagents behaved as active-site-directed irreversible inhibitors of the enzyme. Bromopyruvate exhibited a high affinity to the enzyme. Iodoacetate (anionic reagent) had a higher affinity than iodoacetamide (neutral reagent).