Genomic organization and chromosomal distribution of rat ID elements.

Identifier (ID) elements are members of a family of short interspersed nuclear elements (SINEs) in rodents. We investigated the genomic organization and chromosomal distribution of the ID elements in the rat, mouse and Chinese hamster. Southern blot hybridization analysis revealed that the ID elements are widespread in the rat genome, but concentrated in the mouse and Chinese hamster genomes, and that the copy of ID elements in the rat is about 5 times and 50 times that in the mouse and Chinese hamster, respectively. FISH analysis showed that the ID elements are predominantly distributed in the R-band regions of rat chromosomes. In mouse and Chinese hamster chromosomes, no specific distribution pattern of the ID elements was found. Furthermore, we found a distinct group of derivative ID elements in the rat, which contain partially repeated ID core domains, by PCR amplification using an ID core sequence. Such derivatives were not found in either the mouse or Chinese hamster. These findings suggest that explosive amplification of the ID elements in the rat has been accompanied by the occurrence of derivative ID elements and a predominant localization to the R-band regions. Similar associations found in the Alu family, one of the human SINEs, allow us to speculate that the rat ID elements and the human Alu family have analogous functions in chromosomal organization.

Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line.

BACKGROUND: Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types. RESULTS: We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice. CONCLUSIONS: The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.

A simple, two-color fluorescence detection method for membrane blotting analysis using alkaline phosphatase and horseradish peroxidase.

We have developed a one-step, two-color fluorescence detection method using simultaneously two fluorogenic substrates for both Southern and Western blots on nylon membranes. For this enzyme-mediated reporter system, a mixture of (i) 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP), a substrate for alkaline phosphatase and (ii) N-(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB), a fluorogenic substrate for horseradish peroxidase was used. The reaction with these substrates produces blue (HNPP) and yellow (AMMB) fluorescent signals under ultraviolet light (302 nm). Therefore, this simple method allows the simultaneous visualization of two different targets on a single nylon membrane, e.g. nucleic acids or proteins.

Simultaneous visualization of Q-bands and FISH signals using a novel fluorochrome.

We developed a new HNPP-azo dye method for detection of fluorescence in situ hybridization (FISH) signals on Q-banded chromosomes by use of a newly synthesized fluorochrome, HNPP (3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamide phosphate ester), which reacts enzymatically with alkaline phosphatase and azo dye. The FISH staining method permits simultaneous detection of orange HNPP signals on chromosomal sites labeled by Q-banding, allowing the assignment of small (440-1,200 bp) probes.

Isolation of Acinetobacter calcoaceticus from the gastrointestinal tracts of SCID mice.

Acinetobacter calcoaceticus colonization was observed in the gastrointestinal tracts of C.-B17-scid/scid (SCID) mice, while it was not observed in C.B17-scid/+ and C.B17-+/+ mice with normal immunity housed under the same conditions. A. calcoaceticus and other viable enteric bacteria were not isolated from any organs other than gastrointestinal tract in SCID mice. The mice colonized with this organism were apparently healthy and no significant visceral lesion was observed.

Characterization of mycoplasma strains isolated from house musk shrews (Suncus murinus) and their infectivity in mice and rats.

Five mycoplasma strains isolated from house musk shrews (Suncus murinus) in the Central Institute for Experimental Animals were characterized and compared with three murine mycoplasma strains, Mycoplasma pulmonis m 53, M. arthritidis PG6, and M. neurolyticum Type A, and with reference strain G3-5 previously isolated from a house musk shrew. These isolates fermented glucose, but did not hydrolyze urea and arginine, passed through membrane filters of 450 nm pore size, were sensitive to digitonin, and formed minute (115 to 231 microns in diameter) colonies on agar medium. All the five unclassified house musk shrew mycoplasma strains and strain G3-5 used as a reference constituted a homogeneous group based on (i) their antigenic properties (determined using the metabolism inhibition test), (ii) their polypeptide profiles (determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blotting assay), and (iii) their genomic properties (determined using DNA cleavage pattern analysis), but were quite distinct from the established murine mycoplasmas on the basis of these findings. In a survey of 56 house musk shrews aged 2 to 45 weeks in our colonies, mycoplasmas were isolated from the oral cavities of all animals examined. No gross or microscopic lesions were observed in the five animals from which the mycoplasma strains were isolated. In experimental infection, the mycoplasma was not infective for mice and rats. The results suggest that this group of mycoplasmas is a common inhabitant of house musk shrews.

Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction.

Sequence analysis of the nucleocapsid protein genes of five strains of mouse hepatitis virus (MHV) disclosed that the 3' region of the nucleocapsid protein gene contains highly conserved sequences unique to MHV. We designed a pair of primers to amplify cDNA from such sequences of MHV by using the polymerase chain reaction (PCR). Six isolates of wild-type MHV, as well as prototype viruses, were amplified successfully and detected in ethidium bromide-stained agarose gels. The sequence identity of PCR products was readily verified by confirming target size and a MflI site within the target. The sensitivity of our PCR assay was estimated to be sufficient to detect a single cell infected with MHV. This new approach may permit more sensitive and rapid detection of MHV in biologic materials than current methods such as virus isolation, the infant mouse bioassay, and the mouse antibody production test.

Application of dried whole blood collected on filter paper disks to ELISA for the detection of Sendai virus and mouse hepatitis virus antibodies in mice.

This study was undertaken to simplify the preparation procedures for test specimens by applying whole blood collected on filter paper disks. The results of ELISA obtained using specimens collected in this way for the detection of Sendai virus and mouse hepatitis virus antibodies in mice were comparable to those for ordinary ELISA using serum samples.

Propagation of CAR bacillus in artificial media.

CAR bacillus propagated successfully in an artificial medium, and the number of CAR bacillus was about 30 times the original number after 8 days of cultivation. The medium consisted of Eagle's minimum essential medium supplemented with 10% fetal calf serum and 20% hamster tracheal organ culture soup. By intranasal inoculation to mice, two strains of the CAR bacillus passaged 5 and 6 times in this artificial medium produced the same lung lesions as natural CAR bacillus infection.

Pathogenesis of CAR bacillus in rabbits, guinea pigs, Syrian hamsters, and mice.

Cilia-associated respiratory (CAR) bacillus isolated from infected mice (designated, CBM) and propagated in embryonated chicken eggs was inoculated intranasally in rabbits (Oryctolagus cuniculus), guinea pigs (Cavia porcellus), hamsters (Mesocricetus auratus) and mice (Mus musculus). Gross and microscopic lesions, localization of CBM antigen in the respiratory tract, development of antibody, and ability to reisolate the CAR bacillus were studied in animals killed at 2-, 4-, or 8-week intervals postinoculation (PI). In rabbits, although no histopathological changes were observed in the respiratory tract, CBM antigen was detected on the ciliated epithelium of the respiratory tract, and serum CBM antibody was also detected 4 and 8 weeks PI. In guinea pigs, no histopathological changes were noted, CBM antigen was detected in the respiratory tract 2 and 4 weeks PI but not 8 weeks PI, and serum CBM antibody was detected 4 and 8 weeks PI. In hamsters, mononuclear cell proliferation in the submucosa of the bronchus and trachea was observed 8 weeks PI. CBM antigen was detected at first in the nasal cavity 2 weeks PI and in the lower respiratory tract 4 and 8 weeks PI and serum CBM antibody was detected 4 and 8 weeks PI. In mice, histopathological changes, CBM antigen and CBM antibody were observed. CBM was reisolated from the tracheal washouts of hamsters and mice 8 weeks PI but not from those of rabbits and guinea pigs. These results confirm and extend previous reports of experimentally-induced CAR bacillus infection in mice, guinea pigs, and rabbits. To this list of susceptible laboratory animals, we now add hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

A serological survey on Bacillus piliformis infection in laboratory rabbits in Japan.

A total of 544 rabbit sera obtained from 6 commercial breeding facilities and 9 research institutions during 1985-1990 were tested for Bacillus piliformis antibody by an enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody test (IFAT). The antibody was detected in 53 (14.2%) rabbits from 3 breeding facilities and 30 (17.4%) rabbits from 6 research institutions, indicating the prevalence of B. piliformis infection among laboratory rabbits in Japan. The overall agreement with ELISA for immune status was 96.9% (527/544) with IFAT. In tests of the ability of ELISA and IFAT to quantitate antibody, a correlation coefficient (r) of 0.86 (P less than 0.01) was obtained by plotting the measured average log of the ELISA titer against the corresponding log of the IFAT titer.

Detection of mouse hepatitis virus antibody by protein A-ELISA in 6 prevalent inbred strains or outbred stocks of mice.

Protein A was applied as a reagent for the secondary reaction in ELISA (protein A-ELISA). Mouse hepatitis virus antibody in 6 prevalent mouse strains or stocks reared in a MHV-contaminated room was effectively detected by protein A-ELISA, whereas significant strain differences in the antibody detection rate were demonstrated using the complement fixation test. C57BL/6 mice were particularly reactive in the protein A-ELISA test.

Determination of an antigen suitable for enzyme-linked immunosorbent assay of the antibody to Bordetella bronchiseptica in guinea pigs.

To establish an enzyme-linked immunosorbent assay (ELISA) technique for the serological diagnosis of infections caused by Bordetella bronchiseptica (B. bronchiseptica) in guinea pigs, the authors recently assessed the usefulness of three antigen preparations derived from the bacterial cell components: sonication antigen (S-Ag), cell surface antigen (C-Ag) and lipopolysaccharide antigen (L-Ag). The use of S-Ag for ELISA resulted in the most sensitive detection of the antibody to B. bronchiseptica from guinea pig sera immunized with killed bacteria and sera derived from naturally infected guinea pigs. Like C-Ag, S-Ag was highly specific, showing no cross-reactivity with Pasteurella multocida. Assessment of antibody formations in animals with experimentally induced infection using the three antigen preparations revealed that the antibody to S-Ag was formed earlier than antibodies to the other two antigen preparations following growth of the bacterium in the lungs. These results indicate that ELISA with S-Ag as an antigen is a useful tool for the serological diagnosis of infection by B. bronchiseptica.

Differences in susceptibility to peroral inoculation with Bacillus piliformis spores in rats and mice.

The median liver lesion producing doses of peroral inoculation with the spores of Tyzzer's organism RJ strain were 10(4. 3) in rats and 10(2. 7) in rats receiving prednisolone treatment for the provocation of Tyzzer's disease. In contrast to rats, liver lesions were detected in few mice inoculated perorally with 10(7) spores. In mice inoculated perorally with 10(7) spores, excretion of infective spores in the feces was detected only on day 1 postinoculation. On the other hand, no difference in susceptibility between rats and mice was detected upon intravenous inoculation with vegetative cells of the RJ strain. These results suggest that germination of the spores in the intestinal tract causes the difference in the susceptibility in rats and mice.

In vitro and in vivo induction of squamous cell carcinoma antigen (SCC) in a uterine cervical cancer cell line (SKG-IIIa) with peplomycin and sodium butyrate.

In order to investigate the effect of an antisquamous cell carcinoma drug, peplomycin, the new analogue of bleomycin, on the production of a squamous cell carcinoma-associated tumor marker termed "SCC" (or TA-4), we carried out in vitro and in vivo experiments using the uterine cervical epidermoid cancer cell line SKG-IIIa, together with the investigation of the effect of sodium butyrate which was reported to be one of the representative gene modulators. In vitro production of SCC was biochemically and immunocytochemically confirmed in SKG-IIIa cells. Immunocytochemistry using anti-SCC antibody revealed that the total number of SCC-positive cells increased after the treatment with peplomycin (1.6 fold) or sodium butyrate (1.5 fold). The total amount of SCC in cultured medium, intracellular SCC, and cell debris during 5 days of culturation also increased with peplomycin (1.8 fold) and sodium butyrate (1.4 fold). These data strongly suggest that SCC production of SKG-IIIa cells is stimulated by peplomycin and sodium butyrate in vitro. In vivo experiments were also performed by administering peplomycin to nude rats with heterotransplanted tumors of SKG-IIIa, and transient elevations of serum SCC level (113% to 238% of the initial values) were observed, suggesting that SCC production of cancer cells is also stimulated by peplomycin in vivo.

Sensitive detection of Mycoplasma pulmonis by using the polymerase chain reaction.

Detection of Mycoplasma pulmonis was examined by using the polymerase chain reaction (PCR) for amplifying a specific DNA sequence. In gel electrophoresis which was conducted to detect the amplified products, only 1 pg of M. pulmonis DNA could be detected following 30 cycles of amplification, while no amplified product was detected even from 1 microgram of M. arthritidis or M. neurolyticum DNA. Furthermore, 10 colony-forming units of M. pulmonis could be detected by direct amplification from the mycoplasma suspension. These results suggest the usefulness of the PCR as a highly sensitive, specific, and rapid method for direct detection of M. pulmonis.

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests.

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.

Studies on the development of an ELISA kit for microbiological monitoring. 2. Improvement of the prototype ELISA kit with special references to mouse hepatitis virus antigen.

Improvement of the mouse hepatitis virus (MHV) antigen in a prototype ELISA kit was performed. Equivalent divalent antigens of MHV Nu-67 and S strains with a protein concentration of 10 micrograms/ml showed the best sensitivity and specificity for the detection of MHV and sialodacryoadenitis/Parker's rat coronavirus antibodies in mice and rats, respectively. An increase in the reliability of macroscopic evaluation of both antibody tests is expected by using the newly manufactured kit with the improved antigen.