Changes of oligosaccharides and fatty acids in monkey hippocampus by synaptic potentiation.

We measured the release of free fatty acids and structural changes of glycoprotein glycans induced by tetraethylammonium (TEA) salt in hippocampal slices of cynomolgus monkey brain. The release of free fatty acids in the hippocampal slices occurred after synaptic potentiation by TEA in a different manner from rat hippocampus. Arachidonic acid release in monkey hippocampus occurred much faster than that in rat. Several types of glycans of monkey hippocampal glycoproteins were determined depending on the duration time after TEA treatment. 5-Mannose was increased within 2 min, while polysialoglycans were increased after 5 min or later. Comparative study of glycans of monkey and rat hippocampal slices revealed the presence of relatively larger amount of sialo- and multi-anntenary glycans in rat than in monkey. These results indicate that the depolarizing stimulation of monkey hippocampal slices induced the change of glycoprotein glycan structures and release of free fatty acids in a different manner from rat hippocampus.

Microbiological contamination in genetically modified animals and proposals for a microbiological test standard for national universities in Japan.

The Biosafety Committee of the Japanese Association of Laboratory Animal Facilities of National Universities (JALAN) investigated recent episodes of microbiological contamination in genetically modified mice (GMM), and the countermeasures taken when the contaminated GMM were introduced into animal facilities, by questionnaires addressed to 53 animal facilities belonging to JALAN and serological tests. Although almost all of the contaminated GMM were accepted with conditions such as rederivation after or before reception and housing in designated rooms, contamination with a spectrum of microorganisms was demonstrated in GMM transferred domestically and from abroad. In serological tests, Mycoplasma pulmonis, mouse parvovirus, and mouse encephalomylitis virus were detected in GMM transferred from domestic facilities and from abroad. The present results of the questionnaires and serological tests suggest that GMM are highly and widely contaminated with microorganisms compared with mice from commercial breeders. Thus, we propose a microbiological requirement, including microbiological status--excellent, common, and minimum--as a guide for the transfer and procurement of mice and rats in Japan.

Prevalence of herpes B virus antibody in nonhuman primates reared at the National University of Japan.

A serological investigation by means of an enzyme immuno assay test for herpes B virus (cercopithecine herpesvirus 1) was performed on 961 sera of healthy nonhuman primates reared in laboratory animal facilities which belong to the Association of Laboratory Animal Facilities of the National University of Japan. An antibody prevalence of 40% (384/ 961) was demonstrated. The antibody titer was shown to be higher among macaques (60% of cynomolgus monkeys, 53% of rhesus monkeys, and 34% of Japanese monkeys) than among non-macaque species (21%). These data indicate that nonhuman primates reared in animal facilities may present an occupational health problem and a potential zoonotic biohazard as demonstrated in limited cases in the United States.

Acetylcholine reactivates latent pseudorabies virus in mice.

The latency model of pseudorabies virus (PrV) wild strain, YS-81, in mice was established and latent PrV reactivated with acetylcholine. The latent PrV was reactivated from the trigeminal ganglia with acetylcholine. It was found that this model is useful in investigating the mechanism of latent PrV reactivation by acetylcholine.

Four conductance levels of cloned cardiac L-type Ca2+ channel alpha1 and alpha1/beta subunits.

Formation of single channel subconductance is one of the unique characteristics of the L-type Ca2+ channel. Although alpha1 subunit exhibits a primary function of the channel, it remains uncertain whether alpha1 subunit alone is able to produce subconductance. We tested this by studying single channel currents of cloned cardiac alpha1 subunit expressed in Chinese hamster fibroblast cells, with/without coexpression of cardiac beta subunit. The alpha1 subunit exhibited four distinct levels of conductance (22.7, 14.3, 6.2 and 3.2 pS). Coexpression of beta subunit significantly increased the number of openings in all four levels of conductance without changing the conductance values.

Alpha-galactosidase transgenic mouse: heterogeneous gene expression and posttranslational glycosylation in tissues.

We produced six transgenic mouse lines expressing human alpha-galactosidase (alpha-Gal) in order to evaluate its posttranslational modification. Among them, serum alpha-Gal activity increased 3000-fold in two transgenic mouse lines (TgN2 and TgN51), as compared to that in non-transgenic lines. The heart and liver of the TgN2 mouse expressed a high amount of transcript as well as high alpha-Gal activity. Its gene products in the heart and kidney were sensitive to endoglycosidase H digestion, but those in the spleen and liver were largely resistant. Glycopeptidase F treatment confirmed an identical molecular mass for the peptide moiety of the enzyme. We concluded that heterogeneous molecular mass of the gene products was caused by different degrees of posttranslational glycosylation in murine tissues.

Perceptions of sexual harassment: the effects of gender, legal standard, and ambivalent sexism.

This research tests the possibility that the reasonable woman as compared to the reasonable person test of hostile work environment sexual harassment interacts with hostile and benevolent sexist beliefs and under some conditions triggers protectionist attitudes toward women who complain of sexual harassment. We administered to a sample of undergraduates the ambivalent sexism inventory along with the fact patterns in two harassment cases and asked them to make legally relevant decisions under either the reasonable woman or person standard. We found that those high in hostile sexism, and women, found more evidence of harassment. However, those high in benevolent sexism did not exhibit the hostile sexism effects. Although men were less sensitive to the reasonable woman standard than women, under some conditions the reasonable woman standard enabled both genders to find greater evidence of harassment. The results are discussed from the perspectives of law and psychology.

Acetylcholine activates latent pseudorabies virus in pigs.

Pseudorabies virus (PrV) was isolated from the nasal swabs and the cultured trigeminal ganglia of latently infected pigs after they were treated with acetylcholine (ACH). These results indicate that ACH activates latent infections of PrV.

Rabies virus M protein expressed in Escherichia coli and its regulatory role in virion-associated transcriptase activity.

Rabies virus M protein was expressed in Escherichia coli in the form of a fusion protein with maltose binding protein (MBP) and purified by amylose affinity column chromatography after extraction. In order to investigate the possible regulatory role of M protein in viral transcription, an assay system for rabies virion-associated transcriptase activity was established by using the ribonucleoprotein (RNP) cores prepared from purified virions. Analysis of the products of the transcription assay system showed that the products are sensitive to RNase and are positive-strand RNA. Addition of the fusion protein to the system after cleavage with a proteinase Factor Xa (FXa), which cleaves the fusion protein into the M protein and MBP, resulted in an efficient and dose-dependent inhibition of the transcription. Furthermore, addition to the system of anti-M protein monoclonal antibody significantly restored the transcription. Control experiments with the same transcription assaying system using rabies virus nucleoprotein expressed as a fusion protein with MBP and cleaved with FXa did not result in an inhibition of the transcription. These results suggest that the M protein of rabies virus has the property to down-regulate virion-associated transcription.

Protective efficacy in mice of post-exposure vaccination with vaccinia virus recombinant expressing either rabies virus glycoprotein or nucleoprotein.

Mice vaccinated intraperitoneally (i.p.) with 10(7) p.f.u. of a vaccinia virus recombinant expressing either the glycoprotein (rVac-G) or nucleoprotein (rVac-N) of rabies virus 3 weeks before challenge were protected against peripheral lethal infection. Similarly, by post-exposure vaccination in which mice were first infected with rabies virus and subsequently vaccinated i.p. with the recombinant, rVac-G conferred protection when given immediately following infection and up to 24 h after infection. Prior treatment of those mice with anti-CD8 monoclonal antibodies (MAb) did not significantly affect the outcome of the infection. In contrast, rVac-N failed to confer protection even with higher doses (10(8) p.f.u.) of the virus or even when administered by the intradermal route. Anti-nucleoprotein antibody production by these mice was not suppressed by prior rabies virus infection and the levels and the time of antibody production were similar to those of anti-glycoprotein antibody production in mice vaccinated with rVac-G after rabies virus infection. The cytotoxic T lymphocyte response was also not down-regulated by rabies virus in the mice that were given rVac-N. Possible mechanism(s) for the ineffectiveness of rVac-N by post-exposure vaccination in contrast to pre-exposure vaccination was discussed.

Nucleotide sequence of the nucleoprotein gene of the RC.HL strain of rabies virus, a seed strain used for animal vaccine production in Japan.

By using a phage vector (lambda ZAP II) and the mRNA extracted from IMR-32 cells infected with the RC.HL strain of rabies virus, we constructed a cDNA library from which four nucleoprotein (N)-specific cDNA clones were obtained by Southern blot hybridization. These clones contained a cDNA insert of about 1.4 kb, in which the longest open reading frame was the same length as that reported for the N cDNA of three fixed strains, CVS, PV, and SAD B19. When the nucleotide and deduced amino acid sequences were compared between the RC.HL and the three strains, homology was within the range of 91.5-91.8% and 95.1-96.0%, respectively. Of 183 nucleotides of the RC.HL N-cDNA that were not identical to that of the corresponding site of at least one of the three strains, 41 were shared with the CVS strain, whereas only three were shared with either of the other two strains. In the amino acid sequence, we found 29 residues that were not shared in common with all of the four strains, 11 of which were the substitutions with radically different amino acids that might cause conformational changes of the protein, and, in addition, five of which were located in the region close to the C terminus. The number of such amino acid substitutions between the RC,HL and CVS strains was smaller than that of the other three strains. These results are not inconsistent with the presumption that the RC.HL and CVS strains originated from the same laboratory strain of the Pasteur viruses.

Target cells of cytotoxic T lymphocytes directed to the individual structural proteins of rabies virus.

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with beta-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.

A unique mutation of glycoprotein gene of the attenuated RC-HL strain of rabies virus, a seed virus used for production of animal vaccine in Japan.

Although the RC-HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.

Linear and conformation-dependent antigenic sites on the nucleoprotein of rabies virus.

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies-related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus-specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2-mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation-dependent epitopes, respectively.

Comparative sequence analysis of the M gene among rabies virus strains and its expression by recombinant vaccinia virus.

Nucleotide sequences and the deduced amino acid sequences of the gene encoding the matrix (M) protein of the Nishigahara and the CVS strains of rabies virus have been determined. The M gene is 609 nucleotides long and is capable of coding for a peptide composed of 202 amino acids. Sequence comparison of these M genes with those of other stains [Pasteur (PV), ERA, Avol] revealed that there is 89.7-91.5% homology at the nucleotide level, and 90.1-92.1% homology at amino acid level, between almost all combinations of these strains. However, in the combinations of the PV and ERA strains, and the virulent CVS and the avirulent CVS-derived Avol strains, much higher homology was observed both at the nucleotide and amino acid levels. The predicted secondary structure and hydropathy profiles also exhibited similar features. Recombinant vaccinia virus containing the M gene was constructed. Sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis of the precipitates obtained by immune reaction of the recombinant virus-infected cell lysate with a monoclonal antibody against the M protein revealed that electrophoretic mobility of the expressed protein is indistinguishable from that of the authentic M protein from rabies virions.

Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection.

Mice were vaccinated with recombinant vaccinia virus (rVac) expressing the glycoprotein (G), nucleoprotein (N), phosphoprotein (NS) or matrix protein (M) of rabies virus and their resistance to peripheral lethal infection with street rabies virus was examined. Mice vaccinated with rVac-G or rVac-N developed strong antibody responses to the corresponding proteins and essentially all mice survived challenge infection. Mice vaccinated with rVac-NS or rVac-M developed only a slight antibody response, however, a significant protection (59%) was observed in the rVac-NS-vaccinated mice, whereas rVac-M-vaccinated mice were not protected. No anti-G antibodies were detected in the sera of mice which has been vaccinated with rVac-N or rVac-NS and survived challenge infection. Passive transfer of anti-N monoclonal antibodies (MAbs) recognizing an epitope located on amino acids 1-224 of the protein prior to challenge resulted in significant protection, although the protection was not complete even with a high amount of antibodies. In contrast, none of the mice given MAbs recognizing an epitope of amino acids 247-415 or F(ab')2 fragments from a protective MAb IgG were protected. Administration of anti-CD 8 MAb to rVac-N-vaccinated mice showed no significant effect on protection. Our observations suggest that a considerable part of the protection achieved by the vaccination with rVac-N can be ascribed to the intact anti-N antibodies recognizing an epitope located on amino acids 1-224 of the protein.

Mapping of the antigenic determinants recognized by monoclonal antibodies against the M2 protein of rabies virus.

Twenty-one hybridomas producing monoclonal antibodies (moAbs) against the M2 protein of the Nishigahara (RECH) strain of rabies virus were prepared using the SDS-polyacrylamide gel-purified M2 protein as the immunogen. All moAbs reacted with the protein after Western blotting of rabies virus. By combinations of competitive binding assays, examination of the reactivity of moAbs to the cells infected with parent RCEH and two other strains, CVS and HEP-Flury, and immunoprecipitation with in vitro translation products derived from full-length and truncated cDNAs of the M2 gene, these moAbs could be classified into seven epitope groups. Of these, 20 moAbs belonging to six epitope groups were suggested to recognize an antigenic determinant in the amino-terminal region, from the 1st to the 72nd amino acid of the protein (8 moAbs from two groups directed to amino acids 1 to 72; 2 moAbs from a group directed to amino acids 9 to 72; 5 moAbs from a group directed to amino acids 17-72; 5 moAbs from two groups directed to amino acids 32 to 72). The antigenic determinant recognized by the remaining 1 moAb was shown to be located in the amino acid region from 50 to 171. These moAbs should be useful for further studies on the biological functions of the M2 protein of rabies virus.

Suppression of cell-mediated immunity by street rabies virus infection.

An attempt to define a severe suppression of cell-mediated immunity by street rabies virus infection was undertaken by using the mice lethally and peripherally infected with a street rabies virus (1088 strain). The cell-mediated cytotoxic (CMC) activity of the spleen cells from those mice once slightly increased until day 4 after infection but declined rapidly thereafter until their death on days 10 to 12 after infection. In parallel with a decrease of CMC response of the spleen cells from 1088-infected mice, proliferative response to Con A, IL-2 activity in the culture supernatants of Con A-induced proliferation, responsiveness to exogenously added IL-2 and to Con A to express IL-2R, of those cells became suppressed, and the marked decrease of the total number of spleen cells was observed. Selective depletion of CD4+ and CD8+ cells in the spleens, abnormalities of IL-1 and E-type prostaglandins (PGE2) production or production of inhibitory component able to block IL-2 activity by spleen cells were not observed and these factors did not appear to be associated with the suppression of proliferative response to Con A. However, an apparent association of CD8+ cells in the suppression of differentiation of pre-cytotoxic lymphocytes (CTL) into CTL was demonstrated in the co-culture experiments of the spleen cells from 1088-infected mice with spleen cells of mice infected with an attenuated rabies virus (ERA strain) which can induce higher levels of CMC response. There was no evidence of the productive replication of rabies virus in thymus and spleen of 1088-infected mice. The relationship of these observations to current theories on virus-induced immunosuppression was discussed.

Conserved nucleotide sequence of rabies virus cDNA encoding the nucleoprotein.

The cDNAs of rabies virus (the CVS strain) encoding the structural proteins (G, N, NS, and M) were cloned. Of these clones, the nucleotide sequence of the cDNA encoding the nucleoprotein was determined to compare with those of other strains of rabies virus. The comparison confirmed that the nucleotide sequences and deduced amino acid sequences are highly conserved among strains including an avirulent strain.