Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 1. Design of benzoheterocyclic analogues

The N'-benzoyl group of N-tert-butyl-N'-benzoyl-3,5-dimethylbenzohydrazide (1) was converted to a series of benzoheterocyclecarbonyl groups in order to investigate the potential usefulness of superimposing a hydrazine insecticide on 20-hydroxyecdysone. A series of analogues with benzodioxole, benzodioxane, benzodioxapine, indole, benzoxazole, benzoxazine or benzothiazole instead of the phenyl group of (1) were synthesized and tested for their insecticidal activity against the common cutworm (Spodoptera litura F). N-tert-Butyl-N'-(3,5-dimethylbenzoyl)-1,3-benzodioxole-5-carbohydrazide and N-tert-butyl-N'-(3,5-dimethylbenzoyl)-2,3-dihydro-1,4-benzodioxine-6-carbohydrazide showed high insecticidal activities, superior to that of (1) and equal to that of the commercial insecticide tebufenozide (RH-5992).     

Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 2. Introduction of substituents on the benzene rings of the benzoheterocycle moiety

A series of N'-benzoheterocyclecarbonyl-N-tert-butyl-3,5-dimethylbenzohydrazide analogues possessing a variety of substituents on the benzene rings of the benzoheterocyle moieties were synthesized and tested for their insecticidal activity. The introduction of a methyl group at the R1 position of the benzoheterocycle moiety strongly increased the insecticidal activity. Among the analogues synthesized, N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-5-methyl-6-chromanecarbohydrazide showed the highest insecticidal activity (LC50 = 0.89 mg litre(-1)).     

Identification and characterization of Marek's disease virus serotype 1 (MDV1) ICP22 gene product: MDV1 ICP22 transactivates the MDV1 ICP27 promoter synergistically with MDV1 ICP4

A previous report [Virus Genes 6 (1992) 365-378] has shown that the US1 gene of Marek's disease virus serotype 1 (MDV1) encodes a homologue of herpes simplex virus type 1 infected cell protein No. 22 (ICP22). In the present study, we expressed and identified a product of the MDV1 US1 gene in chicken embryo fibroblasts (CEFs) with the aid of a recombinant baculovirus expressing a Flag epitope-tagged MDV1 US1 gene, under control of the SRalpha promoter (composed of the enhancer region of the simian virus 40 early promoter and the R region of the human T-cell leukaemia virus type 1 long terminal repeat). In CEF infected with the recombinant baculovirus, MDV1 ICP22 was specifically and efficiently expressed in the presence of n-butyric acid. The apparent M(r) of the expressed protein was 30,000. Reporter gene assays revealed that MDV1 ICP22 by itself transactivated an MDV1 ICP27 promoter/reporter construct weakly but specifically, and furthermore, worked synergistically with MDV1 ICP4 to efficiently up-regulate the MDV1 ICP27 promoter. MDV1 ICP22 may be a regulatory protein that stimulates viral promoters in co-operation with other viral regulatory proteins such as MDV1 ICP4.     

芽孢杆菌JPC-2的营养竞争测定及其对小麦根部病害的防治效果

对营养竞争拮抗测定方法进行了改进,对接法将芽孢杆菌JPC-2与小麦纹枯病菌接种于大豆麦麸(SWB)培养基上,JPC-2可在SWB培养基上迅速扩展并完全抑制小麦纹枯病菌的菌丝生长。田间试验表明,芽孢杆菌JPC-2液体菌剂(107cfu/mL,1∶100)处理种子对小麦纹枯病的冬前期防效高于扬花期防效,防治效果为61.9%,高于3%苯醚甲环唑包衣处理(用量为种子质量的0.3%);对小麦根腐病的防治效果可达46.8%,与空白对照差异显著(p<0.05);并使小麦增产73.5%,高于3%苯醚甲环唑种衣剂。芽孢杆菌JPC-2固体菌剂(106cfu/mL,225kg/hm2)沟施于土壤,再播种液体菌剂包衣(107cfu/mL,药种比1∶30)的小麦种子,对小麦纹枯病菌的冬前期防效为68.9%,扬花期防效为59.7%,小麦增产38.9%,与0.2%戊唑醇种衣剂(1∶50)增产效果相当。     

Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat

ABSTRACT. To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.     

Evaluation of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent antibody (IFA) test for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibody in pigs from conventional farms

To evaluate the immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA) for detecting the porcine reproductive and respiratory syndrome virus (PRRSV) antibody, conventional pigs in PRRSV-positive and -negative commercial farms were examined. Antibody development patterns in ELISA and IFA tests were compared in 3 week old piglets experimentally infected with the PRRSV. The virus was detected from 2 days post infection (PI) and then the antibody titers and S/P ratios rose by both methods. A total of 208 serum samples were collected from 4 PRRSV-negative farms and 210 samples from PRRSV-positive farms, and were tested for the PRRSV antibody by IFA and ELISA. The titer of 64 should be set as the cut-off point in IFA for field sera. Similarly, the cut-off S/P ratio should be set at 0.4 in ELISA. A high degree of correlation was observed between antibody titers by the two methods in these 418 samples, with a correlation coefficient of 0.84. The coincidence rate between the two tests was 84.7% (354/418). In non-coincident cases, ELISA was able to detect the antibody with a low titer in the serum samples which were negative in IFA but from PRRSV positive farms. ELISA was more sensitive than IFA to detect PRRSV infected animals or farms.     

Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium

Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals. Germ-free animals, however, enable to study response against infection without background effect of other microorganisms. Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA. In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups. TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection. In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella. These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis.     

Rapid detection of chitosanase activity in chitosanase gene-transformed strains of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> by lytic infection of specific bacteriophages

 In combination with lytic infection by virulent phages, a simple method for monitoring transgenic strains of Enterobacter cloacae was developed in this study. First, 15 strains of E. cloacae were used as indicator bacteria to isolate virulent phages with different host ranges. Of the phages isolated, five isolates (EcP-22, -35, -45, -55, and -70) were used to construct a set of virulent phages corresponding to all strains of E. cloacae. Using this phage set, a rhizosphere strain (KRM-055E) of E. cloacae was effectively screened from field soil. KRM-055E was transformed with a prokaryotic chitosanase gene csnSM1 and infected with the phage EcP-03, which can lyse the strain most effectively. The lysis of KRM-055E/csn occurred 2 h after inoculation, and the chitosanase activity was simply detected by dropping the lysate onto an agar plate containing glycol chitosan. The positive signal for chitosanase activity was detected in the 2-h lysates, and the signal intensity reached a maximum in the 5-h lysate. The present assay was simple, rapid, inexpensive, easy to perform, and applicable to another strains. Received: August 2, 2002 / Accepted: October 31, 2002 Acknowledgments This work was supported in part by a grant (no. 99L01205) from the “Research for the Future” program of the Japan Society for the Promotion of Science. We are grateful to Dr. M. Sato, National Institute of Agrobiological Science, Dr. H. Okamoto, Fukui Agricultural Experiment Station, and Dr. K. Tsuda, Kyoto Prefectural Institute of Agricultural Biotechnology, for kindly providing E. cloacae strains. We thank Dr. P. Park, Kobe University, for technical support with the electron microscopic observations.     

Partial Purification of Thai Isolate of Citrus Huanglongbing (Greening) Bacterium and Antiserum Production for Serological Diagnosis

We aimed to improve the purification of citrus Huanglongbing (greening) bacterium (HB), Candidatus Liberobacter asiaticum and to produce an antiserum against HB. Periwinkle plants Catharantus roseum L. graft-inoculated with HB were used to produce an antiserum. All young leaves of new shoots incubated at 20–25°C and 25–30°C, a few mature leaves incubated at 20–25°C, and all mature leaves incubated first at 25–30°C and later transferred to 20–25°C developed yellowing symptoms and were then used to prepare immunogen. The HB was partially purified from these leaves by an improved method that included a macerating enzyme treatment of the midribs of infected leaves and homogenization of infected phloem sieve tissues. An antiserum raised against partially purified HB reacted clearly at a dilution of 1/16 with HB-infected citrus extract prepared at a concentration of 40 times, but did not react with healthy or tristeza virus-infected citrus extract in microprecipitin tests. Received 23 August 2002/ Accepted in revised form 4 December 2002