11 research outputs found
A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH
Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis
Genetic Analysis of Noroviruses in Chiba Prefecture, Japan, between 1999 and 2004
Noroviruses (NVs) are common pathogens that consist of genetically divergent viruses that induce gastroenteritis in humans and animals. Between September 1999 and June 2004, 1,898 samples obtained from patients showing sporadic or outbreak gastroenteritis in Chiba Prefecture, Japan, were tested for NVs by reverse transcription-PCR. NVs were detected in 603 samples. Approximately 80% were positive for genogroup GII, 13% were positive for genogroup GI, and the remaining 7% were positive for both genogroups. Phylogenetic analysis showed that the GI and GII genogroups could be further divided into 13 and 16 genotypes (including new genotypes), respectively. The GII-4 genotype, which included five small genetic clusters (subtypes), was the most common in this study and was detected in approximately 40% of positive samples. The P2 regions of 10 strains belonging to each of the five GII-4 subtypes showed 5 to 18% amino acid diversity. The amino acid substitutions accumulated in the protruding (P) region during the 5-year study period. Our data suggest that highly variable NV strains are circulating in Chiba Prefecture, with a high rate of genetic change observed during the 5-year study period
Characterization of processing pores and their relevance to the strength in alumina ceramics
Molecular Cloning, Expression, and Antigenicity of Seto Virus Belonging to Genogroup I Norwalk-Like Viruses
The viral capsid protein of the Seto virus (SeV), a Japanese strain of genogroup I Norwalk-like viruses (NLVs), was expressed as virus-like particles using a baculovirus expression system. An antigen detection enzyme-linked immunosorbent assay based on hyperimmune antisera to recombinant SeV was highly specific to homologous SeV-like strains but not heterologous strains in stools, allowing us type-specific detection of NLVs
Microtubule reorganization during the cell cycle in synchronized BY-2 tobacco suspensions
Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells
Chlorinated Naringenin Analogues as Potential Inhibitors of Transthyretin Amyloidogenesis
Misfolding and aggregation of transthyretin
are implicated in the
fatal systemic disease known as transthyretin amyloidosis. Here, we
report the development of a naringenin derivative bearing two chlorine
atoms that will be efficacious for preventing aggregation of transthyretin
in the eye. The amyloid inhibitory activity of the naringenin derivative
was as strong as that of tafamidis, which is the first therapeutic
agent targeting transthyretin in the plasma. X-ray crystal structures
of the compounds in complex with transthyretin demonstrated that the
naringenin derivative with one chlorine bound to the thyroxine-binding
site of transthyretin in the forward mode and that the derivative
with two chlorines bound to it in the reverse mode. An ex vivo competitive
binding assay showed that naringenin derivatives exhibited more potent
binding than tafamidis in the plasma. Furthermore, an in vivo pharmacokinetic
study demonstrated that the dichlorinated derivative was significantly
delivered to the eye
Chlorinated Naringenin Analogues as Potential Inhibitors of Transthyretin Amyloidogenesis
Misfolding and aggregation of transthyretin
are implicated in the
fatal systemic disease known as transthyretin amyloidosis. Here, we
report the development of a naringenin derivative bearing two chlorine
atoms that will be efficacious for preventing aggregation of transthyretin
in the eye. The amyloid inhibitory activity of the naringenin derivative
was as strong as that of tafamidis, which is the first therapeutic
agent targeting transthyretin in the plasma. X-ray crystal structures
of the compounds in complex with transthyretin demonstrated that the
naringenin derivative with one chlorine bound to the thyroxine-binding
site of transthyretin in the forward mode and that the derivative
with two chlorines bound to it in the reverse mode. An ex vivo competitive
binding assay showed that naringenin derivatives exhibited more potent
binding than tafamidis in the plasma. Furthermore, an in vivo pharmacokinetic
study demonstrated that the dichlorinated derivative was significantly
delivered to the eye