Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrP(Sc)) has generated the potential for sensitive detection of prions. Here we developed a new PrP(Sc) amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrP(Sc) in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD.     

Vaccine efficacy of Mycobacterium bovis BCG against Mycobacterium sp. infection in amberjack Seriola dumerili

Mycobacteriosis, caused by the intracellular parasitism Mycobacterium sp., causes economic damages to aquaculture production in Japan, particularly in seriola fish production. Antibiotics are not effective against Mycobacterium sp. and so a potent vaccine is needed. We previously reported that BCG vaccine (Mycobacterium bovis BCG) induces adaptive immunity against Mycobacterium sp. in Japanese flounder, Paralichthys olivaceus. In a phylogenetic tree, the genes for a major antigen, the Ag85 complex, in Mycobacterium sp. TUMSAT-Msp001 are closely related to homologues in Mycobacterium ulcerans. M. bovis BCG was detected until 7 days post-injection at the injection site (muscle) and 28 days post-vaccination in spleen. Cumulative mortality of amberjack, Seriola dumerili vaccinated intramuscularly (i.m.) and intraperitoneally (i.p.) with M. bovis BCG was 32.3% and 59.5% respectively, at 24 days post-infection of Mycobacterium sp., compared to 97.8% in PBS-injected fish. The bacterial counts of Mycobacterium sp. in spleen of both i.m.-and i.p.-vaccinated fish (6.2 × 10³ and 1.3 × 10⁴ CFU/mg tissue, respectively) at 20 days post-infection were significantly lower (P < 0.01) than those of PBS-injected fish (8.0 × 10⁶ CFU/mg). Furthermore, Immersion challenge with Mycobacterium sp. TUMSAT Msp-001 showed 50% RPS value in BCG i.m.-vaccinated fish at the end of the experiment. These results support our previous study using Japanese flounder and suggest that BCG vaccine is also effective against Mycobacterium sp. infection in amberjack.     

Uncovering the Mechanisms of Shrimp Innate Immune Response by RNA Interference

Because of the importance of shrimp in world aquaculture, there is much interest in understanding their immune system in order to improve their resistance to pathogenic microorganisms. An effective tool in studying genes involved in the immune response in shrimp is RNA interference (RNAi). RNAi, first recognized as an antiviral response against RNA viruses, is a cellular mechanism that is triggered by double-stranded RNAs and results in the degradation of homologous genes. In this review, we describe the current studies of genes in shrimp that employed RNAi technology to elucidate or confirm their functions. We also review the potential of RNAi to elicit antiviral response in shrimp.     

Ascorbic acid induces a marked conformational change in long duplex DNA.

Ascorbic acid is often regarded as an antioxidant in vivo, where it protects against cancer by scavenging DNA-damaging reactive oxygen species. However, the detailed mechanism of the action of ascorbic acid on genetic DNA is still unclear. We examined the effect of ascorbic acid on the higher-order structure of DNA through real-time observation by fluorescence microscopy. We found that ascorbic acid generates a pearling structure in single giant DNA molecules, with elongated and compact regions coexisting along a molecular chain. Results from electron microscopy and atomic force microscopy indicate that the compact regions assume a loosely packed conformation. A possible mechanism for the induction of this conformational change is discussed in relation to the interplay between the higher-order and second-order structures of DNA.     

New RNAi strategy for selective suppression of a mutant allele in polyglutamine disease

In gene therapy of dominantly inherited diseases with small interfering RNA (siRNA), mutant allele specific suppression may be necessary for diseases in which the defective gene normally has an important role. It is difficult, however, to design a mutant allele-specific siRNA for trinucleotide repeat diseases in which the difference of sequences is only repeat length. To overcome this problem, we use a new RNA interference (RNAi) strategy for selective suppression of mutant alleles. Both mutant and wild-type alleles are inhibited by the most effective siRNA, and wild-type protein is restored using the wild-type mRNA modified to be resistant to the siRNA. Here, we applied this method to spinocerebellar ataxia type 6 (SCA6). We discuss its feasibility and problems for future gene therapy.     

Identification of three isoforms for mitochondrial adenine nucleotide translocator in the pufferfish Takifugu rubripes

Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.     

siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.