The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Antigenic analysis of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13

In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced. All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes. To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E. coli. Western blotting analysis showed that there are at least four major ASs on PO-13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24). Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain. MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays. MAbs against AS I reacted with turkey strains, Ty-1 and Ty-3, but not with a chicken strain, Ch-1. Nine of 11 MAbs against AS II cross-reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes. These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.     

Genetic characterization of rabies field isolates from Thailand

We sequenced 512 nucleotides in two variable regions of the N gene of 23 rabies isolates from the northeastern part of Thailand by direct sequencing of PCR-amplified products. The sequencing data revealed two new lineages in these rabies isolates. Based on the results of this study together with the findings of our earlier study, the rabies isolates in Thailand were divided into two genogroups, designated as T1 and T2, which were predominantly localized in the northern and northeastern areas, respectively. Each of these two genogroups consisted of four lineages. There was a correlation between these eight lineages and the geographical origins of the isolates. Two lineages belonging to the T2 genogroup from the northeastern area of Thailand were newly identified in this study. The isolates in both genogroups were also prevalent in the central area of Thailand. Each lineage in the T1 and T2 genogroups was found independently in dogs in the upper and lower southern areas of Thailand, respectively. These genetic data and the historical background suggest that rabies viruses belonging to the T2 genogroups were prevalent many years ago in the central and northeastern areas of Thailand and were later transferred to the lower southern parts of Thailand.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo

Pathogens or pathogen-associated molecular patterns can signal to cells of the innate immune system and trigger effective adaptive immunity. However, relatively little is known about how the innate immune system detects tissue injury or necrosis. Evidence suggests that the release of heat-shock proteins (HSPs) may provide adjuvant-like signals, but the ability of HSPs to promote activation or tolerance in vivo has not been addressed. In this study we show that Hsp70 promotes dendritic cell (DC) function and, together with antigen, triggers autoimmune disease in vivo.     

Convenient method for determining the absolute configuration of chiral alcohols with racemic 1H NMR anisotropy reagent,M(alpha)NP acid: use of HPLC-CD detector

A convenient method for determining the absolute configuration of chiral secondary alcohols using the racemic NMR anisotropy reagent, (+/-)-2-methoxy-2-(1-naphthyl)propionic acid [(+/-)-M(alpha)NP acid], and an HPLC-CD detector was developed. The method was successfully applied to some chiral alcohols derived from (-)-alpha-santonin.     

Periplasmic secretion of native ovalbumin without signal cleavage in Escherichia coli

In Escherichia coli cells carrying wild-type ovalbumin cDNA, some of the recombinant protein was secreted into the periplasmic space. In contrast, a signal-region mutant form of ovalbumin (deletion, Gly1 to Ala39) was not detected in the periplasm despite being synthesized at the same level as the wild-type protein. Chemical and spectroscopic analyses showed that periplasmic ovalbumin assumes a conformation indistinguishable from that of native egg white ovalbumin. We concluded that a process resembling the secretion of ovalbumin process in the oviduct occurs also in bacteria.