Thymine starvation-induced structural changes in Escherichia coli DNA. Detection by pulsed field gel electrophoresis and evidence for involvement of homologous recombination.

Effect of thymine starvation on Escherichia coli DNA was investigated by using pulsed field gel electrophoresis combined with cell lysis in agarose gel. Post-lysis treatment with restriction enzymes generating relatively large fragments (NheI, SpeI or XbaI) revealed peculiar electrophoretic profiles specific for thymine-starved cells. Thus, a substantial portion of the DNA remained in the origin of electrophoresis (non-migrating DNA), and the amounts of the migrating fragments correspondingly decreased in an inverse relation to the map distance between the origin of replication (oriC) and each fragment. The formation of non-migrating DNA seems to depend upon the presence of replicated portions of the chromosome (sister duplexes), as judged by the effect of a preincubation at the non-permissive temperature in a dnaA(Ts) mutant. Electron microscopy showed that the non-migrating fraction of DNA was enriched with such structures as single-stranded tails or gaps and branchings with single-stranded arms. It was also found that the appearance of non-migrating DNA was highly dependent on the functional recA gene and moderately on certain RecF-family genes. These results strongly suggest that homologous recombination between sister duplexes is involved in the formation of the peculiar structures found in non-migrating DNA. A possible causal relationship between the formation of non-migrating DNA and viability loss (thymineless death) is also discussed.

The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation.

A 2,695 bp chromosomal segment of Escherichia coli K12 containing the recQ gene was sequenced. Analysis of the sequence revealed an open reading frame thought to represent recQ, with a clockwise direction of transcription relative to the standard genetic map of E. coli K12 and having a coding capacity for a protein of Mr 68,350. The -10 region of the presumptive recQ promoter overlapped the putative terminator for the upstream gene pldA, and was immediately followed by a 15 bp stretch of DNA bearing a strong resemblance to the reported sequences of LexA repressor binding sites. This latter finding suggested the possibility of SOS regulation of recQ gene expression, which was substantiated by experiments with recQ-lacZ fusions.

The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants.

The recQ gene of Escherichia coli K12 was subcloned from plasmid pKO1 (Oeda et al. 1981) by monitoring the capacity of the resulting recombinant plasmids partially to reverse the increased ultraviolet (UV) sensitivity of a recF143 recQ1 double mutant. We were able to trace this complementation activity to a 3.4 kilobase (kb) SalI-PvuII fragment. Furthermore, analysis of the Tn3 insertion mutations that abolished the complementation revealed the exclusive localisation of such insertions in the same 3.4 kb segment. This segment was situated about 4 kb clockwise from corA on the chromosome, a result consistent with the transductional data previously reported. In addition, a comparison of our restriction endonuclease cleavage map with the published data has placed recQ between pldA and pldB. When relocated to the recQ site on the chromosome, the recQ::Tn3 mutations conferred partial resistance to thymineless death (TLD) or, in the case of a recBC sbcB background, recombination deficiency and increased UV sensitivity. This has provided the firm evidence that both the TLD resistance and the deficiency in the RecF recombination pathway result from loss of the functional recQ gene. We also identified the recQ gene product as a 74 kilodalton polypeptide by using the maxicell technique.

recA+ gene-dependent regulation of a uvrD::lacZ fusion in Escherichia coli K12.

The expression of the Escherichia coli uvrD gene was studied with a uvrD::Mud(Aprlac) insertion mutant. The results indicate that it is inducible by DNA damaging agents in a recA+ gene-dependent manner.

Post-harvest degradation of carotenoid glucose esters in saffron.

It has been found that an indoor cultivation system of Crocus sativus L. is more favorable with regard to the quality of saffron, as compared to the usual cultivation in an open field. Carotenoid glucose esters increase from the period before blooming and reach the maximum in the full blooming period, and are sensitive for the presence of oxygen, light irradiation, and beta-glucosidase. Moreover, it is evident that storage of saffron at -20 degrees C promotes the constant supply of saffron with a homogeneous pharmacological activity.

Glycosidic constituents from in vitro Anoectochilus formosanus.

The glycosidic constituents of whole plants of Anoectochilus formosanus propagated by tissue culture were investigated. A new compound, 2-(beta-D-glucopyranosyloxymethyl)-5-hydroxymethylfuran, along with the known compounds, 3-(R)-3-beta-D-glucopyranosyloxybutanolide (kinsenoside), 3-(R)-3-beta-D-glucopyranosyloxy-4-hydroxybutanoic acid, 1-O-isopropyl-beta-D-glucopyranoside, (R)-(+)-3,4-dihydroxy-butanoic acid y-lactone, 4-(beta-D-glucopyranosyloxy)benzyl alcohol, (6R,9S)-9-hydroxy-megastigma-4,7-dien-3-one-9-O-beta-glucopy ranoside, and corchoionoside C were isolated.

Hepatoprotective aliphatic glycosides from three Goodyera species.

Hepatoprotective aliphatic glycosides 3-(S)-3-beta-D-glucopyranosyloxybutanolide (1) and its congener, 3-(S)-3-beta-D-glucopyranosyloxy-4-hydroxybutanoic acid (2) were isolated as major constituents from the whole plants of three Goodyera species, G. schlechtendaliana Reichb. fil., G. matsumurana Schltr. and G. discolor Ker-Gawl. The structures of 1 and 2 have been determined by NMR, MS spectroscopic and chemical means. Compound 1 was converted into its methyl ester form (5) during the purification step, when the lactone ring was cleaved by catalysis of silica gel with the CHCl3-MeOH-H2O as a solvent. On the other hand, 1 was obtained in a high yield by the same purification procedure without MeOH. Based on this fact, a simple and economic method for the purification of 1 was confirmed. Compounds 1 and 2 were found to have a hepatoprotective effect on liver injury induced by carbon tetrachloride in primary cultured rat hepatocytes.

Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway.

An Escherichia coli K12 mutant resistant to thymineless death (TLD) was isolated, and its genetic analysis led us to identify a new mutation (recQ1) located between corA and metE on the standard linkage map. The mutation was found to result in increased sensitivity to ultraviolet light and deficiency in conjugational recombination when placed in the recBC sbcB background, indicating that it blocked the RecF pathway of recombination. It seemed likely that this mutation is also capable of causing partial resistance to TLD, but we reserve the possibility of a separate mutation closely linked to recQ1 giving rise to this phenotype. The original mutant was shown to carry an additional mutation probably in the vicinity of the uhp locus, which was also required for the full TLD resistance of the mutant to be expressed.

Higher yielding isolation of kinsenoside in Anoectochilus and its antihyperliposis effect.

A higher concentration of kinsenoside, 3-(R)-3-beta-D-glucopyranosyloxybutanolide (1), was detected in the crude drug Anoectochilusformosanus, and A. koshunensis by HPLC analysis. A methylation reaction occurred to give methyl ester (4) when the lactone ring of 1 was cleaved by silica gel catalysis using methanol containing solvent used in the purification step resulting in difficulty to purify 1. To avoid the cleavage reaction, a reversed-phase or silica gel column without methanol was used to give a high yield of 1. In an anti-hyperliposis assay using high-fat diet rats, 1 significantly reduced the weights of body and liver, and also decreased the triglyceride level in the liver compared to those of control rats. On the other hand, the epimer of 1, 3-(S)-3-beta-glucopyranosy-loxybutanolide (2), trivially named goodyeroside A, which was isolated from Goodyera species, had no effect for anti-hyperliposis. In aurothioglucose-induced obese mouse, 1 suppressed the body and liver weight increase, significantly ameliorated the triglyceride level in the liver, and also reduced the deposition of uterine fat-pads.

Inhibitory effects of cucurbitane triterpenoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumor. II.

To search for possible anti-tumor-promoters, we carried out a primary screening of twenty-four 29-nor-cucurbitacin glucosides isolated from the roots of Cayaponia tayuya (Cucurbitaceae) using an in vitro synergistic assay system. Of these glucosides, cayaponosides B (5), B3 (7), D (8), D3b (22) and C2 (23) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, 5 and 23 exhibited remarkable anti-tumor-promoting effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Strong antinociceptive effect of incarvillateine, a novel monoterpene alkaloid from Incarvillea sinensis.

Incarvillea sinensis is a wild plant distributed in northern China. The dried whole plant has been traditionally used to treat rheumatism and relieve pain as an ancient Chinese crude drug. To investigate its antinociceptive activity, we evaluated several fractions derived from the methanolic extract of Incarvillea sinensis in the formalin-induced pain model in mice. Incarvillateine, a novel monoterpene alkaloid, has been found to show significant antinociceptive activity. Here we report the antinociceptive activity of incarvillateine and compare its activity with that of morphine. Additionally, we suggest that its action may be related to influence on the central opioid pathways.