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     

Reduction of Spore Density of Plasmodiophora brassicae in Soil by Decoy Plants

The severity of clubroot (Plasmodiophora brassicae) on Chinese cabbage was reduced by growing plants such as oats, spinach and leafy daikon prior to Chinese cabbage in pot experiments. Resting spore densities of P. brassicae in the soil were 29–62%, depending on the pervious crop, as compared to unplanted control plot after ploughing under the previously cultivated plants. Root hairs of the preceding plants were infected with P. brassicae, but clubbed roots were not formed on these plants. The results indicate that these plants functioned as decoy plants reducing the resting-spore density in soil and thereby suppressing disease severity. Received 21 February 2000/ Accepted in revised form 5 September 2000     

Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.