Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae.

UNLABELLED: Erysipelothrix rhusiopathiae is a causative agent of swine erysipelas. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of E. rhusiopathiae. The LAMP assay correctly detected 39 E. rhusiopathiae strains. No LAMP products were detected from 14 non-rhusiopathiae Erysipelothrix and 16 non-Erysipelothrix strains, including E. tonsillarum serovar 10 strains, which are difficult to be discriminated from E. rhusiopathiae strains. These results were consistent with those obtained by a conventional E. rhusiopathiae-specific PCR assay. Starting with DNA extraction from a single colony, the gel-based PCR assay took 4 h to provide a result, but the LAMP assay was faster, requiring only 37-80 min. The conventional culture test required more than 3-4 days to isolate and identify E. rhusiopathiae in the enrichment cultures. In contrast, the LAMP assay required less than 22 h from the beginning of the enrichment culture to final determination. These results suggest that the LAMP assay is useful as an adjunct to facilitate early diagnosis of swine erysipelas. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a loop-mediated isothermal amplification (LAMP) assay for simple and cost-effective detection of E. rhusiopathiae from swine samples. The LAMP assay provided more rapid detection of the bacterium than conventional PCR and biochemical-based assays, and it may potentially facilitate surveillance and early diagnosis of swine erysipelas in the field.

Pathogenicity of Erysipelothrix rhusiopathiae: virulence factors and protective immunity.

Erysipelothrix rhusiopathiae is the causative agent of erysipelas in animals and erysipeloid in humans. In the absence of specific antibodies, the organism evades phagocytosis by phagocytic cells, but even if phagocytized, it is able to replicate intracellulary in these cells. In this review, recent advances in our understanding of the pathogenicity of E. rhusiopathiae and its protective immunity are described.

Porcine caspase-3: its cloning and activity during apoptosis of PK15 cells induced by porcine Fas ligand.

We cloned and sequenced cDNA that contained the coding sequence of porcine caspase-3. The open reading frame (ORF) of porcine caspase-3 cDNA was 834 base pairs (bp) in length and encoded 277 amino acids. The predicted amino acid sequence was 88.4%, 86.6%, and 87.7% homologous to the predicted human, murine, and rat amino acid sequences, respectively. The activity of caspase-3 in porcine renal tubular cell line PK15 after recombinant porcine Fas ligand (FasL) stimulation was examined. The enzymatic activity of caspase-3, but not that of caspase-1, was significantly increased after FasL treatment. Western blot analysis also showed that the processing of caspase-3 from proenzyme to mature subunits occurred after FasL treatment. The inhibition of caspase-3 by its specific inhibitor partially prevented the apoptotic cell death of PK15 cells caused by FasL. The porcine caspase-3 cDNA isolated in this study will be useful for the study of apoptotic cell death in pigs and will lead to the discovery of therapeutic uses of caspases and their inhibitors in the prevention of viral and bacterial diseases and tissue injury associated with xenotransplantation and allotransplantation.

Molecular cloning of porcine interleukin-1beta converting enzyme and differential gene expression of IL-1beta converting enzyme, IL-1beta, and IL-18 in porcine alveolar macrophages.

We have cloned and sequenced a cDNA that contains the coding sequence of porcine interleukin-1beta (IL-1beta) converting enzyme (ICE). Using degenerate oligonucleotide primers based on the amino acid sequences of the human, murine, and rat ICE, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine alveolar macrophages stimulated with lipopolysaccharide (LPS) to clone the cDNA of porcine ICE. The open reading frame (ORF) of the porcine ICE cDNA is 1215 base pairs (bp) in length and encodes 404 amino acids. The predicted amino acid sequence is 72.5%, 62.6%, and 64.1% homologous to the human, murine, and rat amino acid sequences, respectively. The kinetics of mRNA expression of ICE, IL-1beta, and IL-18 in porcine alveolar macrophages after LPS stimulation revealed that ICE transcripts were weakly expressed in nonstimulated condition and upregulated after LPS stimulation. Moreover, IL-1beta and IL-18 transcripts were differently expressed after LPS stimulation.

Efficient production of biologically active porcine interleukin-18 by coexpression with porcine caspase-1 using a baculovirus expression system.

We previously reported that the precursor form of porcine interleukin-18 (IL-18) expressed by the baculovirus system was able to be secreted efficiently into the supernatant of insect cells, whereas only small amounts of mature IL-18 were secreted from insect cells. As insect cells do not normally have the IL-1beta converting enzyme (caspase-1), which is required for processing of the precursor IL-18 into the mature IL-18, we recently cloned porcine caspase-1 cDNA. In this study, we constructed a recombinant baculovirus containing the cDNA encoding porcine caspase-1 and showed that the coexpression of caspase-1 and the precursor IL-18 enabled insect cells to secrete mature IL-18 into the culture supernatant efficiently. Moreover, inhibition of caspase-1 activity by its specific inhibitor prevented the processing of precursor IL-18 into the mature form. These results indicated that the processing and secretion of precursor IL-18 into the mature form in insect cells were enhanced by the artificial introduction of caspase-1 activity for cleavage.

Molecular cloning, characterization, and expression of porcine Fas ligand (CD95 ligand).

We isolated and sequenced cDNA that contained the coding sequence of porcine Fas ligand (FasL). Using mixed oligonucleotide primers based on the 5' and 3' nucleotide sequences conserved among human, murine, and rat FasL, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine thymocytes stimulated with 5 microg/ml concanavalin A (ConA) to clone the cDNA of porcine FasL. The open reading frame (ORF) of porcine FasL cDNA was 849 base pairs (bp) in length and encoded 282 amino acids. The predicted amino acid sequence was 85.5%, 76.6%, and 75.5% homologous to the predicted human, murine, and rat FasL, respectively. The recombinant porcine FasL expressed by recombinant baculovirus containing the whole coding sequences of porcine FasL showed cytotoxic effect and induced apoptosis in porcine renal tubular cell line PK-15 cells sensitized by cycloheximide (CHX), which was confirmed by MTT assay, DNA fragmentation assay, and TUNEL staining, respectively. Furthermore, the mRNA expression of porcine FasL in porcine peripheral blood lymphocytes (PBL) was induced by porcine interleukin-18 (IL-18). These results indicate that porcine FasL identified in this study is biologically functional and has the ability to induce apoptosis as reported in other species.

Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies.

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.

Production of monoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purification of recombinant porcine interleukin-18.

We have recently reported the cloning and expression of porcine interleukin-18 (IL-18). In this study, we describe the production of anti-porcine IL-18 monoclonal antibodies (mAb) and their use in the purification of a large amount of recombinant porcine IL-18 by immunoaffinity column chromatography. Five monoclonal antibodies (2-2-B, 2-5-B, 2-13-C, 3-1-C and 5-3-B) were established and characterized. Three (2-2-B, 3-1-C and 5-3-B) of them were of IgG1 subclass, and the other two were IgMs. Epitope analysis of the three IgG1 mAbs showed that they recognized the same epitope. All five mAbs demonstrated reactivity with baculovirus generated porcine IL-18 by immunoblot analysis. Biologically active porcine IL-18 was obtained by immunoaffinity chromatography using anti-porcine IL-18 mAb at more than 85% purity from culture supernatants of Trichoplusia ni (Tn5) derived cells infected with recombinant baculovirus containing the coding sequence of porcine mature IL-18. These results suggest that the anti-porcine IL-18 mAbs established in this study are useful for one-step purification of porcine mature IL-18 as well as the detection of porcine IL-18 by immunoblotting.

Synthesis and biological action of the aminotetrahydroisoquinocarbazoles and related compounds: a new class of compounds with antiarrhythmic activity.

A series of 12-aminotetrahydroisoquinocarbazoles and related compounds were synthesized using an intramolecular Diels-Alder reaction and screened for antiarrhythmic activity in chloroform-induced ventricular arrhythmias in mice. Several compounds showed more potent activity than disopyramide. There was some correlation between substituents on aromatic ring and angular position, and antiarrhythmic activity. An amino group or some functional groups containing an amino group on C-12 seemed to be essential to exhibit the activity. Ring size also influenced the activity. The compound (+)-10 (RS-2135) had the most favorable combination of antiarrhythmic activity and toxicity and was selected for further evaluation.

Studies on cardiovascular agents. 6. Synthesis and coronary vasodilating and antihypertensive activities of 1,2,4-triazolo[1,5-a]pyrimidines fused to heterocyclic systems.

The synthesis and coronary vasodilating and antihypertensive activities of 1,2,4-triazolo[1,5-a]pyrimidines fused to pyrrole, thiophene, pyran, pyridine, and pyridazine are described. Among these compounds, 8-tert-butyl-7,8-dihydro-5-methyl-6H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-a]pyrimidine (23) was found to be the most promising potential cardiovascular agent, having been shown to be more potent in coronary vasodilating activity than trapidil [7-diethylamino)-5-methyl-1,2,4-triazolo[1,5-a]pyrimidine] and approximately equipotent to guanethidine sulfate in antihypertensive activity.

Nonpeptide angiotensin II receptor antagonists: synthesis, biological activities, and structure-activity relationships of imidazole-5-carboxylic acids bearing alkyl, alkenyl, and hydroxyalkyl substituents at the 4-position and their related compounds.

A series of imidazole-5-carboxylic acids bearing alkyl, alkenyl, and hydroxyalkyl substituents at the 4-position and their related compounds were prepared and evaluated for their antagonistic activities to the angiotensin II (AII) receptor. Among them, the 4-(1-hydroxyalkyl)-imidazole derivatives had strong binding affinity to the AII receptor and potently inhibited the AII-induced pressor response by intravenous administration. Various esters of these acids showed potent and long-lasting antagonistic activity by oral administration. The most promising compounds were (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (CS-866) and (pivaloyloxy)-methyl esters of 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[(2'-1H-tetrazol-5- ylbiphenyl-4-yl)-methyl]imidazole-5-carboxylic acid (26c). A study involving stereochemical comparison of 26c with the acetylated C-terminal pentapeptide of AII was also undertaken.

Synthesis and biological evaluation of novel tricyclic carbapenems (trinems).

Synthesis of new tricyclic carbapenems (trinems) with a pyrrolidinyl moiety at the C-4 position of the tricyclic ring and their antimicrobial activities were studied. These trinems showed potent activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Among them, (4R)-[(S)-pyrrolidin-3-ylthiomethyl]trinem (14a) exhibited good activity against MRSA in vitro and in vivo.

Synthesis and antibacterial activity of novel 1beta-methyl carbapenems with cycloalkylamine moiety at the C-2 position.

Novel 1beta-methyl carbapenems with a cycloalkylamine moiety as a side chain were synthesized and their structure-activity relationships were studied. These carbapenems showed potent antibacterial activities against a wide range of Gram-positive and Gram-negative bacteria, and moderate urinary recovery when administered intraperitoneally in mice.

Extract of vinegar "Kurosu" from unpolished rice inhibits the proliferation of human cancer cells.

The effects of the ethyl acetate extract of "Kurosu" (EK), Japanese traditional vinegar from unpolished rice, on the proliferation of a variety of human cancer cell lines were investigated by using the alamar blue assay. Cancer cell lines included colon adenocarcinoma (Caco-2), lung carcinoma (A549), breast adenocarcinoma (MCF-7), bladder carcinoma (5637), and prostate carcinoma (LNCaP) cells. EK inhibited the proliferation of all tested cell lines in a dose-dependent manner, with inhibition mostly pronounced in Caco-2 cells (up to 62% inhibition at a dose level of 0.025%). Flow cytometry of EK-treated Caco-2 cells showed a decrease in cell number in the G2/M phase and an increase in the sub-G1 phase (apoptotic). In addition, DNA fragmentation was detected in Caco-2 cells cultured with EK by immunostaining. RT-PCR analysis revealed p21 mRNA expression was induced in EK-treated Caco-2 cells. Moreover, PARP cleavage was promoted in EK-treated Caco-2 cells. These results suggest that EK causes G0/G1 arrest through p21 induction and, thus, is a potential apoptosis inducer in Caco-2 cells.

Extract of vinegar "Kurosu" from unpolished rice inhibits the development of colonic aberrant crypt foci induced by azoxymethane.

The modifying effects of administrating an ethyl acetate extract of "Kurosu" (EK), a vinegar made from unpolished rice, on development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of EK on proliferating cell nuclear antigen (PCNA) index in ACF, prostaglandin (PG) E2 expression in the colonic mucosa and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in the liver. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt). They also received drinking water containing 0, 0.05, 0.1 or 0.2% EK for 4 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 140 +/- 23 ACF/rat at the end of the study (week 4). EK administration dose-dependently inhibited ACF formation and inhibition by 0.2% EK was statistically significant (P < 0.002). Treatment with EK significantly lowered PCNA index in ACF and reduced PGE2 content in the colonic mucosa, while EK elevated liver GST and QR activities. These findings suggest that EK may be effective for inhibiting colonic ACF, through induction of liver GST and QR and possibly alteration of PGE2 production.

Plasma-dependent chemotactic activity of hemocytes derived from a juvenile estuarine gastropod mollusc, Clithon retropictus, to Vibrio parahaemolyticus and Escherichia coli strains.

Hemocytes of adult Clithon retropictus were attracted chemotactically to live Vibrio parahaemolyticus and Escherichia coli strains. The chemotaxis was stimulated by the plasma of adult C. retropictus. Hemocytes of the juvenile specimen were attracted chemotactically to V. parahaemolyticus and E. coli strains in the presence of the plasma of the juvenile and only to E. coli strain in the absence of the plasma. These evidences suggest that hemocytes of juvenile C. retropictus might be defective to recognize V. parahaemolyticus strains and that the hemocytes would display full activities in the presence of the plasma factor(s).

Improved electroporation of Rhodococcus equi.

The condition of an electroporation method was re-evaluated for the introduction of foreign plasmid DNA into Rhodococcus equi. The method is based on an electroporation of the bacteria made competent by culturing in a broth containing glycine and by heat shock at 50 degrees C. Transformation of R. equi could be achieved with a chloramphenicol-resistant shuttle vector originating from Rhodococcus fascians at an efficiency of about 10(4) transformants/microgram DNA. The bacteria were also shown to become competent when they were incubated with a chemical transformation buffer prior to washing with an electroporation buffer.

Proliferative response and cytokine production of bovine peripheral blood mononuclear cells induced by the superantigens staphylococcal enterotoxins and toxic shock syndrome toxin-1.

The potential of staphylococcal enterotoxin A (SEA), B (SEB), C(SEC) and toxic shock syndrome toxin-1 (TSST-1) to act as superantigens by inducing polyclonal T-cell mitogenesis and cytokine production was tested on bovine peripheral blood mononuclear cells (PBMC). These four toxins were capable of inducing strong proliferative response of PBMC from calves over a broad dosage range (1 pg/ml to 1 microgram/ml) in vitro. The toxin-activated blast cells consisted of both CD4+ T-cells and CD8+ T-cells, but the T-cell proliferation depended upon the presence of monocytes. Treatment of monocytes with monoclonal antibody to major histocompatibility complex class II antigens substantially inhibited the toxin-induced T-cell proliferative response, but paraformaldehyde-fixation did not abrogate the accessory function. SEA, SEB, SEC and TSST-1, all induced the in vitro release of interleukin-2, interferon gamma and tumor necrosis factor alpha in a dose dependent manner. The results indicate that SEA, SEB, SEC and TSST-1 are capable of acting as superantigens by stimulating bovine T-cells as shown in the human and murine systems. The possible implications of these toxins in the immunopathogenesis of bovine mastitis caused by the infection with Staphylococcus aureus are discussed.

The influence of pregnancy on sensation of ear problems--ear problems associated with healthy pregnancy.

We wondered how many women had experienced a sensation of fullness in the ear during pregnancy. To address this question, data were obtained from a group of healthy women who attended the gynaecology clinic in our hospital as pregnancy cases between February 1995 and January 1998 and who volunteered to participate in our study. A control group was drawn from healthy female co-medical staff members of our hospital who had never been pregnant. The data used for comparing the two groups were taken from a questionnaire about ear problems that was presented to all subjects. The results suggest that ear problems may be increased in pregnancy, particularly for hypotensive pregnant women. However, even for pregnant women complaining of ear problems, pure-tone audiometry and impedance audiometry showed normal hearing in all cases. Furthermore, these women's complaints resolved completely on delivery of their babies.

[Synthesis and benzodiazepine receptor binding studies of pyridazino[3,4-b]indoles].

A series of methyl 9H-pyridazino[3,4-b]indole-3-carboxylates and related compounds were synthesized using a Diels-Alder reaction of methyl 3-(1H-indol-3-yl)-2-propenoates and dibenzyl azodicarboxylate. Several compounds were found to have high affinity for the benzodiazepine receptor. Their structure-activity relationships are discussed.