The establishment of the food safety commission (FSC) and its role in relation to boiling spongiform encephalopathy (BSE) in Japan

After the detection of the first case of bovine spongiformencephalopathy (BSE) in Japan, severalmeasures were introduced to protect public and animal health. Those measures included BSE testing of all cattle slaughtered for human consumption with a rapid test, removal of specified risk materials (SRM), enhancement of surveillance, and feed ban. In addition, the Food Safety Basic Law was enforced and the Food Safety Commission (FSC) was established in July 2003 to strengthen the function of the government in food safety. In December 2004, the first case of BSE was detected in the United States, and the Japanese government suspended importation of beef from the US to Japan, causing a new trade issue between the two countries. This article outlines how the Japanese government addressed the domestic BSE issues and bilateral trade issues in consultation with the FSC.Après la détection du premier cas d'encéphalopathie spongiforme bovine (ESB) au Japon, plusieurs mesures ont été prises pour protéger la santé publique et animale. Elles comprennent le dépistage de l'ESB, par un test rapide, de tous les bovins abattus pour la consommation humaine, le retrait des matériels à risques spécifiés (MRS), le renforcement de la surveillance et l'interdiction des farines de viandes et d'os. En outre, la Loi fondamentale sur la sécurité alimentaire a été appliquée et la Commission de la sécurité sanitaire des aliments (CSSA) a été créée en juillet 2003 pour conseiller le gouvernement en matière de sécurité alimentaire. En décembre 2004, suite au premier cas d'ESB détecté aux Etats-Unis, le gouvernement japonais a suspendu l'importation de viande bovine qui en provenait, provoquant un nouveau problème commercial entre les deux pays. Cet article décrit la façon dont le gouvernement japonais, après consultation de la CSSA, a contrôlé, au plan national, la situation relative à l'ESB et les relations commerciales bilatérales

The 5′ Flanking Region and Intron1 of the Bovine Prion Protein Gene (PRNP) Are Responsible for Negative Feedback Regulation of the Prion Protein

Transcription factors regulate gene expression by controlling the transcription rate. Some genes can repress their own expression to prevent over production of the corresponding protein, although the mechanism and significance of this negative feedback regulation remains unclear. In the present study, we describe negative feedback regulation of the bovine prion protein (PrP) gene PRNP in Japanese Black cattle. The PrP-expressing plasmid pEF-boPrP and luciferase-expressing plasmids containing the partial promoter fragment of PRNP incorporating naturally occurring single-nucleotide or insertion/deletion polymorphisms were transfected into N2a cells. Transfection of pEF-boPrP induced PrP overexpression and decreased the promoter activity of PRNP in the wild-type haplotype (23-bp Del, 12-bp Del, and −47C). Reporter gene assays further demonstrated that the 12- and 23-bp Ins/Del polymorphisms, which are thought to be associated with Sp1 (Specific protein 1) and RP58 (Repressor Protein with a predicted molecular mass of 58 kDa), in intron1 and the upstream region, respectively, and an additional polymorphism (−47C→A) in the Sp1-binding site responded differently to PrP overexpression. With the −47C SNP, the presence of the Del in either the 23-bp Ins/Del or the 12-bp Ins/Del allele was essential for the negative feedback caused by PrP overexpression. Furthermore, deletion mutants derived from the wild-type haplotype showed that nucleotides −315 to +2526, which include the 5′-flanking region and exon1, were essential for the response. These results indicate that certain negative feedback response elements are located in these sequences, suggesting that regulation by transcription factors such as Sp1 and RP58 may contribute to the negative feedback mechanism of PRNP

Deletion mutants show that the 23-bp deletion in the upstream region of <i>PRNP</i> and/or the 12-bp deletion in intron1, coupled with the absence of the Sp1 SNP and the presence of exon1, are required for the negative feedback response to PrP overexpression during regulation of prion protein expression.

<p>(A) Deletion mutants of the DelDel constructs as shown on the left were used. Dotted box = Luciferase gene; black boxes = exon 1 and exon 2, which include numbers denoting the position of the reported transcription start site (+1) of the <i>PRNP</i> promoter region. The 23-bp indel, 12-bp indel, and SNP regions are also indicated above the reporter gene constructs. The absence (−) and presence (+) of each region in the reporter gene constructs are shown in the Table on the right. (B) Graph representing relative luciferase activities obtained with the above reporter plasmids in the presence of either an empty vector or pEF-boPrP. Relative luciferase activities (Mean ± S.D.) for 3 replicate experiments were compared with the pGL3-control plasmid (1%). A significant difference of luciferase activity in pEF-boPrP-transfected cells as compared with corresponding empty vector-transfected cells is shown by two asterisks (**, <0.01). NS indicates no significant difference.</p

SNP constructs show that the 23-bp deletion in the upstream region of <i>PRNP</i> and/or a 12-bp deletion in intron1, coupled with the absence of the Sp1 SNP and the presence of exon1, are required for the negative feedback response to PrP overexpression.

<p>(A) Map of the portion of bovine <i>PRNP</i> containing the 5′-flanking region and exons 1 and 2 is shown on the top line. Dotted box = Luciferase gene; black boxes = exon 1 and exon 2, which include numbers denoting the position of the reported transcription start site (+1) of the <i>PRNP</i> promoter region. The 23-bp indel, 12-bp indel, and SNP regions are also indicated above the reporter gene constructs. The absence (−) and presence (+) of each region in the reporter gene constructs are shown in the Table on the right. (B) Graph representing the relative luciferase activities obtained with the above reporter plasmids in the presence of either an empty vector, pEF-BOS (EM, open bars), or pEF-boPrP (PrP, solid bars). The pGL3-Control vector (with the standard SV40 promoter) was used for normalization between different experiments (relative light units (<i>RLU</i>) = (firefly luciferase_construct/<i>total protein</i>_construct)/(firefly luciferase_control/<i>total protein</i>_control)). Relative luciferase activities (Mean ± S.D.) for 3 replicate experiments were compared with that of the pGL3-control plasmid (1%). A significant difference of luciferase activity in pEF-boPrP-transfected cells as compared with corresponding empty vector-transfected cells is shown by one asterisk (*, <0.05) or two asterisks (**, <0.01). NS indicates no significant difference.</p

Expression of PrP in pEF-boPrP-transfected N2a cells.

<p>The bovine PrP expression vector pEF-boPrP was transfected into N2a cells. Forty eight hours after transfection, cells were lysed, and then the proteins were separated by 12% SDS-PAGE and blotted. The resulting blots were analyzed using anti-PrP mAb 6H4 as a specific antibody for bovine PrP protein, SAF 32 for PrP of all species, or B-5-1-2 for α-tubulin as an internal control.</p

Expression analyses of HIV-1 Vpr protein in human monocyte-derived macrophages (MDMs).

<p>(A) Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors through leukophoresis, cultured <i>in vitro</i>, and differentiated into MDMs as described in Materials and Methods. At day 7, the MDMs were infected with either Ad-Vpr or Ad-Zs, or were left untreated as mock-infected controls (left). At 48 h post-infection, the cells from Donor 1 were visualized by fluorescence (FL) and bright field phase contrast (BF) microscopy. (B) The cells from the two donors (upper panel, Donor 1; lower panel, Donor 2) were lysed and subjected to Western blot analyses using Vpr, ZsGreen1, and β-actin antibodies.</p

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p

Validation of differentially expressed genes at the protein level.

<p>Human monocyte-derived macrophages (MDMs) were infected with Ad-Vpr or Ad-Zs, or mock-infected as a control. At 48 h post-infection, the cells were washed, lysed, and subjected to Western blot analyses with the indicated antibodies. A β-actin antibody was used as a loading control.</p

Differentially expressed genes (fold change >2.0) associated with immune response (GO: 0006955) upon Ad-Vpr infection in Donor 1 and Donor 2.

<p>Differentially expressed genes (fold change >2.0) associated with immune response (GO: 0006955) upon Ad-Vpr infection in Donor 1 and Donor 2.</p

Gene ontology of differentially expressed genes after infection of human monocyte-derived macrophages (MDMs) with Ad-Vpr.

<p>(A) Venn diagram representing the number of differentially expressed cellular genes (>2-fold change in both donors) after infection of human MDMs with Ad-Vpr. (B) The top ten genes ontology classified by corrected p-value, and (C) heat map of hierarchical gene clustering of the 66 differentially regulated in both donors. Gene up-regulation is denoted in red and gene down-regulation is denoted in blue.</p