Transgenic mice recapitulate the phenotypic heterogeneity of genetic prion diseases without developing prion infectivity: Role of intracellular PrP retention in neurotoxicity

Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome and fatal familial insomnia (FFI). The reason for this variability is not known. It has been suggested that prion strains with unique self-replicating and neurotoxic properties emerge spontaneously in individuals carrying PrP mutations, dictating the phenotypic expression of disease. We generated transgenic mice expressing the FFI mutation, and found that they developed a fatal neurological illness highly reminiscent of FFI, and different from those of similarly generated mice modeling genetic CJD and GSS. Thus transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs expressed in these mice are misfolded but unable to self-replicate. They accumulate in different compartments of the neuronal secretory pathway, impairing the membrane delivery of ion channels essential for neuronal function. Our results indicate that conversion of mutant PrP into an infectious isoform is not required for pathogenesis, and suggest that the phenotypic variability may be due to different effects of mutant PrP on intracellular transport.     

Oligomeric forms of peptide fragment PrP(214-226) in solution are preferentially recognized by PrP(Sc)-specific antibody

A specific monoclonal antibody (mAb) V5B2 that discriminates between brain tissue of Creutzfeldt-Jakob disease patients and that from normal controls without proteinase K digestion has been prepared using a 13-residue synthetic peptide P1 from the primary structure of human PrP. In the light of the specific interaction between mAb V5B2 and the pathological isoform of PrP (PrP(Sc)), we investigated the solution behavior of antigen P1 and its interactions with mAb V5B2. Our results show that V5B2 recognizes epitope P1 in dimeric/oligomeric forms in solution and in the fibril-like aggregates, as well as in PrP(Sc) aggregates, and demonstrate that the specific epitope is present in all of these forms, but not in PrP(C).     

Biological and biochemical characterization of M2B cells: Classical BSE prion is conserved in transgenic mice overexpressing bovine prion protein gene

M2B cells with persistent classical bovine spongiform encephalopathy (C-BSE) have been established previously. In this study, we performed strain characterization of the M2B cell line in bovine PrP^C overexpressing mice (Tg 1896). Mice intracranially inoculated with M2B cells and C-BSE survived for 451 ± 7 and 465 ± 31 d post inoculation, respectively. Although biochemical properties, including deglycosylation and conformational stability, differed between M2B cells and C-BSE, inoculation with M2B cell lysate and C-BSE resulted in comparable phenotypes. Comparable vacuolation scores and PrP^Sc depositions were observed in the brain of Tg 1896 inoculated with both M2B cell lysate and C-BSE. Our results show that biochemical and biological characteristics of M2B cells and C-BSE are classifiable in the same strain.     

Cloning and polymorphism analysis of prion protein gene in domestic bactrian camel in China

Up to now, little is known about the prion protein gene (PRNP) of domestic bactrian camels, and no polymorphisms of the bactrian camel PRNP have been analyzed or reported. In this study, we cloned and analyzed the PRNP sequences of 89 domestic bactrian camels. The results showed that the amino acid sequence of bactrian camel PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of dromedary camels. A four octapeptide PHGGGWGQ repeat region follows a nonapeptide (PQGGGGWGQ) in the N-terminal of deduced amino acid sequence from residues 54 to 95. Polymorphisms of PRNP in both species of camels were observed in codons 16(A → V), 17(M → T), 120(N → S), 176(R → K), 215(I → V), 234(S → Y), 237(Y → S), and 239(Q → G) by comparing with other ruminants. The PrP gene nucleotide sequence alignments of bactrian camels (HQ204566.1 and HQ204567.1) showed high identity with dromedary camel (99.2%, 99.1%), sheep (91.9%, 91.8%) and cattle (91.8%, 91.6%). This study provides valuable data for future research on susceptibility or resistance of camels to prion diseases.     

Identification of diabetes susceptibility loci in db mice by combined quantitative trait loci analysis and haplotype mapping

To identify the disease-susceptibility genes of type 2 diabetes, we performed quantitative trait loci (QTL) analysis in F(2) populations generated from a BKS.Cg-m+/+Lepr(db) and C3H/HeJ intercross, taking advantage of genetically determined obesity and diabetes traits associated with the db gene. A genome-wide scan in the F(2) populations divided by sex and db genotypes identified 14 QTLs in total and 3 major QTLs on chromosome (Chr) 3 (LOD 5.78) for fat pad weight, Chr 15 (LOD 6.64) for body weight, and Chr 16 (LOD 8.15) for blood glucose concentrations. A linear-model-based genome scan using interactive covariates allowed us to consider sex- or sex-by db-specific effects of each locus. For the most significant QTL on Chr 16, the high-resolution haplotype comparison between BKS and C3H strains reduced the critical QTL interval from 20 to 4.6 Mb by excluding shared haplotype regions and identified 11 nonsynonymous single-nucleotide polymorphisms in six candidate genes.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

ER chaperones in mammalian development and human diseases

The field of endoplasmic reticulum (ER) stress in mammalian cells has expanded rapidly during the past decade, contributing to understanding of the molecular pathways that allow cells to adapt to perturbations in ER homeostasis. One major mechanism is mediated by molecular ER chaperones which are critical not only for quality control of proteins processed in the ER, but also for regulation of ER signaling in response to ER stress. Here, we summarized the properties and functions of GRP78/BiP, GRP94/gp96, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, EDEM, Herp and co-chaperones SIL1 and P58(IPK) and their role in development and diseases. Many of the new insights are derived from recently constructed mouse models where the genes encoding the chaperones are genetically altered, providing invaluable tools for examining the physiological involvement of the ER chaperones in vivo.     

Analysis of kinetic data in transport studies: New insights from kinetic studies of Na+-d-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling,rapid filtration apparatus

Summary Using the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991.J. Membrane Biol. 122:111–125), we have studied the kinetic characteristics of Na⁺-d-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35°C using a dynamic approach which involves linearregression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20°C, in the presence of high substrate concentrations or 1mm phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classicalv versus S plot. All together, our results support the existence of a single Na⁺-d-glucose cotransport system in these membranes for which Na⁺ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989.Am J. Physiol. 256:8618–8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters ofd-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved; (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.     

Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens

Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.