A Convenient Synthesis of 4-Hydroxy-3-methyl-2-(2-propynyl)-cyclopent-2-enone,an Alcohol Moiety of a Synthetic Pyrethroid Having Strong Killing and Knockdown Activity

We examined the primary structure of the α-amylase produced by Bacillus subtilis var. amylosacchariticus by attempting to isolate tryptic peptides of the enzyme. By solubilization and precipitation in buffers, the peptides were first fractionated into three. The main fraction was fractionated by ion-exchange chromatography. Twenty-seven peptides were generated from this fraction. The fraction insoluble at neutral pH was fractionated by SP-Sephadex C-25. From this fraction three peptides were obtained. The other fraction insoluble at acidic pH was fractionated by Bio-Gel P-60. Four peptides were isolated from this fraction. In total, thirty-four peptides were generated from the tryptic digest of the α-amylase. The amino acid sequences of twenty-one out of thirty-four peptides were completely determined, while those of the other thirteen peptides were partially determined. The peptides derived from the N- and C-terminal ends of the α-amylase were identified.     

Phyllospadine,a New Flavonoidal Alkaloid from the Sea-Grass Phyllosphadix iwatensis

Attempts to solubilize active ubiquinol: cytochrome c reductase, cytochrome b-c₁ complex, from the submitochondrial particles from sweet potato root tissue ended in failure because all detergents tested caused inactivation of this enzyme complex. Consequently, the complex was isolated with the content of cytochrome b as the marker for purification after solubilization with deoxycholate though it was inactive. Deoxycholate had no effect on two ±-bands at 555 and 558 nm but caused a blue shift of an ±-band at 563 nm in the reduced-minus-oxidized difference spectrum of the submitochondrial particles at low temperature. The purified complex exhibited the same difference spectra at low and room temperatures as the submitochondrial particles in the presence of deoxycholate, which suggests that the complex has three (at least two) cytochrome b components with different spectroscopic properties and that the apparent molar ratio of cytochrome b to c₁ is 1.5. The purified complex consisted of eight subunits: I, 51 kDa; II, 49kDa; III, 33kDa; IV, 32 kDa; V, 27 kDa; VI, 17 kDa; and VII and VIII, 10 kDa. Subunits III and IV were cytochrome c₁ and b, respectively.     

Ceramicines from Chisocheton ceramicus as lipid-droplets accumulation inhibitors

In our search for lipid-droplets accumulation (LDA) inhibitors, some ceramicines, a series of limonoids isolated from the barks of Chisocheton ceramicus, were discovered to be active as anti-LDA. Preliminary structure–activity relationships (SAR) of ceramicines as LDA inhibitors based on ceramicines A–L (1–12) and ceramicine B derivatives were described.     

Cloning and transcriptional analysis of the gene encoding 5-aminolevulinic acid synthase of the white-rot fungus Phanerochaete sordida YK-624

In this study, we cloned the gene encoding 5-aminolevulinic acid synthase (ALAS) from the hyper-lignin-degrading fungus Phanerochaete sordida YK-624. The deduced amino acid sequence showed highest identity (93.0%) to ALAS of P. chrysosporium. Expression of the gene encoding ALAS, which we named aas, corresponded temporally with the expression and activity of manganese peroxidase.     

Isolation, structure identification and SAR studies on thiosugar sulfonium salts, neosalaprinol and neoponkoranol, as potent α-glucosidase inhibitors

Two hitherto missing members of sulfonium salts family in Salacia genus plants as a new class of α-glucosidase inhibitors, neoponkoranol (7) and neosalaprinol (8), were isolated from the water extracts, and their structures were unambiguously identified. For further SAR studies on this series of sulfonium salts, several epimers of 7 and 8 were synthesized, and their inhibitory activities against rat small intestinal α-glucosidases were evaluated. Among them, 3'-epimer of 7 was found most potent in this class of molecules, and revealed as potent as currently used antidiabetics, voglibose and acarbose.     

DNA degradation in an amber mutant of Escherichia coli K12 affecting DNA ligase and viability

Isolation of an amber mutant lig-321 (or dnaL321) if Escherichia coli K12 with a defect in DNA ligase activity was previously reported (Nagata & Horiuchi, 1974). This was the first demonstration that, in E. coli, conditionally lethal nonsense mutants can be isolated selectively. Unlike the hitherto available E. coli K12 DNA ligase-deficient (lig) mutants, the DNA of this mutant is degraded under lethal conditions. This paper describes its further characterization. The DNA degradation was found to be an energy-requiring process, in which endonuclease I did not seem to participate. Kinetic analyses of prelabeled DNA indicated that the parental strands were degraded. The sedimentation profile of prelabeled DNA in an alkaline sucrose gradient showed that the extensive degradation was preceded by a step in which the parental strands were broken into relatively large pieces. At least in the early phase of degradation, which we examined by alkaline sucrose gradient centrifugation of pulse-labeled DNA, synthesis of discontinuous daughter chains (Okazaki fragments, Okazaki et al., 1968) was confirmed. Joining of the nascent chains, however, was completely inhibited. Genetic analyses revealed that the mutant allele is recessive to the wild type. This agrees with in vitro studies in which the mutant crude extract was found not to inhibit DNA ligase activity of the wild type extract. These and other properties of the lig-321 mutant were compared with the other DNA ligase-deficient mutants of E. coli. The role of this enzyme in DNA replication, repair and recombination is discussed.     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.