Acaricidal activity of Thymus vulgaris oil and its main components against Tyrophagus putrescentiae, a stored food mite.

The acaricidal activities of compounds derived from Thymus vulgaris (thyme) oil against Tyrophagus putrescentiae were assessed using an impregnated fabric disk bioassay, and were compared with those of the synthetic acaricides, benzyl benzoate and N,N-diethyl-m-toluamide. The observed responses differed according to dosage and chemical components. The 50% lethal dose (LD50) value of the T. vulgaris oil against T. putrescentiae was 10.2 microg/cm2. Biologically active constituents derived from T. vulgaris oil were purified by using silica gel chromatography and high-performance liquid chromatography. The structures of acaricidal components were analyzed by gas chromatography-mass spectrometry, 1H nuclear magnetic resonance (NMR), 13C NMR, 1H-13C COSY-NMR, and DEPT-NMR spectra, and were subsequently identified as carvacrol and thymol. Carvacrol was the most toxic compound with LD50 values (4.5 microg/cm2) significantly different from thymol (11.1 microg/cm2), benzyl benzoate (11.3 microg/cm2), and N,N-diethyl-m-toluamide (13.9 microg/cm2). Linalool was as toxic as was N,N-diethyl-m-toluamide. The lower LD50 of carvacrol indicates that it may be the major contributor of the toxicity of T. vulagaris oil against the stored food mite, although it only constitutes 14.2% of the oil. From this point of view, carvacrol and thymol can be very useful as potential control agents against stored food mite.

Growth-inhibiting activity of active component isolated from Terminalia chebula fruits against intestinal bacteria.

The growth-inhibitory activity of materials derived from the fruit of Terminalia chebula was evaluated against six intestinal bacteria by means of an impregnated paper disk agar diffusion method. The butanol fraction of T. chebula extract had profound growth-inhibitory activity at a concentration of 5 mg per disk. The biologically active component isolated from the T. chebula fruits was identified with a variety of spectroscopic analyses as ethanedioic acid. The growth responses varied in accordance with the bacterial strain, chemical, and dosage tested. In a test with concentrations of 2 and 1 mg per disk, ethanedioic acid had strong and moderate inhibitory activity against Clostridium perfringens and Escherichia coli, respectively, with no associated adverse effects on the growth of the four tested lactic acid-producing bacteria. Ellagic acid derived from T. chebula fruits exerted a potent inhibitory effect against C. perfringens and E. coli, but little or no inhibition was observed with treatments of behenic acid, P-caryophyllene, eugenol, isoquercitrin, oleic acid, ca-phellandrene, 3-sitosterol, stearic acid, a-terpinene, terpinen-4-ol, terpinolene, or triacontanoic acid. These results may be an indication of at least one of the pharmacological properties of T. chebula fruits.

Mutational study of the role of tyrosine-49 in the Saccharomyces cerevisiae xylose reductase.

The xyl1 gene encoding xylose reductase was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. The purified enzyme readily carried out xylose reduction in vitro. It prefers NADPH as the co-enzyme by about 80-fold over NADH. Compared to the native enzyme purified from S. cerevisiae (Kuhn et al., 1995), the recombinant xylose reductase displayed slightly higher (about two-fold) affinities (K(m)) for the substrate (xylose) and co-factor (NADPH), as well as a 3.9-fold faster turnover number (K(cat)) and 7.4-fold greater catalytic efficiency (K(cat)/K(m)). The reason for the apparent discrepancies in kinetic constants between the recombinant and native S. cerevisiae xylose reductases is not known. Replacement of Tyr49 by Phe in the recombinant enzyme led to greater than 98% loss of activity, suggesting that this residue plays a critical role in catalysis. Intrinsic enzyme fluorescence spectroscopic analysis showed that the wild-type and the Y49F variant both bound the co-enzyme NADPH with similar affinity. This supports the view that Tyr49 is involved in interaction with the substrate and not the co-factor during catalysis.

Identification and Characterization of Acinetobacter sp. CNU961 Able to Grow with Phenol at High Concentrations.

An Acinetobacter sp., strain CNU961, with a higher tolerance to phenol was isolated, and identified through a set of taxonomic studies and a genetic complementation test. Enzymatic and mutagenic studies found that the strain dissimilate phenol by hydroxylation to catechol followed by an ortho-ring cleavage pathway to further mineralize it. The phenol hydroxylase, which is an inducible enzyme and requires NADPH for optimum activity, was not inhibited by phenol at concentrations up to 0.5 mM. The different kinetic behaviors of the enzyme activities on NADPH and on phenol reflected that the phenol hydroxylase of strain CNU961 is a multisubunit allosteric enzyme consisting of heterogeneous polypeptides.

Cloning of catBCIJFD genes for catechol degradation into chromosomal pobA and genetic stability of the recombinant Acinetobacter calcoaceticus.

A possible obstacle in the development of hybrid strains of Acinetobacter calcoaceticus by the introduction of a metabolic pathway into the chromosome is genetic instability of the resulting recombinant strains. Therefore, the possibility that the pobA gene can be used as a chromosomal cloning site where the transposed genes can be maintained and expressed, was explored in this study. For this purpose, two model hybrid strains of A. calcoaceticus were created, in which a DNA fragment carrying catBCIJFD genes for catabolic degradation of catechol was inserted into pobA in opposite directions of each other, and their genetic stabilities were experimentally examined. Our data demonstrated that the stability of the genes neighboring the insertions depends on the orientations of the insertions. Also, the data further indicated that the functional metabolic pathways introduced into pobA can be expressed successfully as far as the insertion is engineered in an appropriate way. Concurrently, it was proposed that the pobA can be used as a chromosomal cloning site, and that introduction of an useful metabolic pathway into pobA may offer considerable promise to the construction of a hybrid strain with improved metabolic capabilities.