Analysis of functional domains of rat mitochondrial Fis1, the mitochondrial fission-stimulating protein

In yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (Dnm1p) in conjunction with a peripheral protein, Mdv1p, and a C-tail-anchored outer membrane protein, Fis1p. In mammals, a dynamin-related protein (Drp1) and Fis1 are involved in the mitochondrial-fission reaction as Dnm1 and Fis1 orthologues, respectively. The involvement of other component(s), such as the Mdv1 homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fis1 (rFis1) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFis1 formed a approximately 200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-down by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFis1. These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFis1 interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFis1.     

Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A(2)alpha

Cytosolic phospholipase A(2) (cPLA(2)) alpha plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA(2)alpha in a Ca(2+)-dependent manner. Peptide microsequencing revealed that purified P60 was identical to vimentin, a major component of the intermediate filament. The interaction occurred between the C2 domain of cPLA(2)alpha and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA(2)alpha and vimentin colocalized around the perinuclear area in cPLA(2)alpha-overexpressing human embryonic kidney 293 cells following A23187 stimulation. Forcible expression of vimentin in vimentin-deficient SW13 cells augmented A23187-induced arachidonate release. Moreover, overexpression of the vimentin head domain in rat fibroblastic 3Y1 cells exerted a dominant inhibitory effect on arachidonate metabolism, significantly reducing A23187-induced arachidonate release and attendant prostanoid generation. These results suggest that vimentin is an adaptor for cPLA(2)alpha to function properly during the eicosanoid-biosynthetic process.     

Pigment exchange of Photosystem II reaction center by chlorophyll d

Pigment exchanges among photosystem reaction centers (RCs) are useful for the identification and functional analysis of chromophores in photosynthetic organisms. Pigment replacement within the spinach Photosystem II RC was performed with Chl d derived from the oxygenic alga Acaryochloris marina, using a protocol similar to that reported previously [Gall et al. (1998) FEBS Lett 434: 88–92] based on the incubation of reaction centers with an excess of other pigments. In this study, we analyzed Chl d-modified monomeric RC which was separated from Chl d-modified dimeric RC by size-exclusion chromatography. Based on the assumption of a constant ratio of two Pheo a molecules per RC, the number of Chl a molecules in Chl d-modified monomeric RCs was found to decrease from six to four. The absorption spectrum of the Chl d-modified monomeric RC at room temperature showed a large peak at 699.5 nm originating from Chl d and a small peak at 672.5 nm orignating from Chl a. Photoaccumulation of the Pheo a⁻ in Chl d-modified monomeric RC, in the presence of sodium dithionate and methyl viologen, did not differ significantly from that in control RC, showing that the Chl d-modified monomeric RC retains its charge separation activity and photochemically active Pheo a.     

Effects of urocortin II on neonatal rat cardiac myocytes and non-myocytes

Urocortin (Ucn) II and III, homologous peptides of Ucn that are specific ligands for corticotropin-releasing hormone (CRH) type 2 receptor (CRH-R2), have recently been identified. The present study was designed to elucidate the effects of Ucn II, which is predominantly expressed in rodent heart, on neonatal rat cardiac myocytes (MCs) and cardiac non-myocytes (NMCs). Ucn II increased the incorporation of [³H]-leucine into MCs, as well as the accumulation of cAMP and the secretion of atrial natriuretic peptide. However, no significant changes were demonstrated in NMCs or an MC/NMC co-culture system. The effects of Ucn II were attenuated by astressin2-B, a specific antagonist of CRH-R2, and/or H89, an inhibitor of protein kinase A (PKA). These results indicate that Ucn II may be another endogenous cardiovascular substance that acts via CRH-R2 and the cAMP-dependent PKA pathway.     

Rapid and quantitative detection of blood Serratia marcescens by a real-time PCR assay: its clinical application and evaluation in a mouse infection model

Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and <10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.     

In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self antigens and induces potent antitumor immunity

Most tumor-associated Ags are self proteins that fail to elicit a T cell response as a consequence of immune tolerance. Dendritic cells (DCs) generated ex vivo have been used to break tolerance against such self Ags; however, in vitro manipulation of DCs is cumbersome and difficult to control, resulting in vaccines of variable potency. To address this problem we developed a method for loading and activating DCs, in situ, by first directing sufficient numbers of DCs to peripheral tissues using Flt3 ligand and then delivering a tumor-associated Ag and oligonucleotide containing unmethylated CG motifs to these tissues. In this study, we show in three different tumor models that this method can overcome tolerance and induce effective antitumor immunity. Vaccination resulted in the generation of CD8(+) T and NK cell effectors that mediated durable tumor responses without attacking normal tissues. These findings demonstrate that unmodified tumor-associated self Ags can be targeted to DCs in vivo to induce potent systemic antitumor immunity.     

Protein/polysaccharide cogel formation based on gelatin and chemically modified schizophyllan

In the work, aldehyde groups were quantitatively introduced into schizophyllan (SPG) side chains through periodate oxidation. The periodate-oxidized SPG (POSPG) forms an elastic gel with gelatin. The cogel formation is based on the Schiff-base reaction between the amino groups of gelatin chains and the aldehyde groups of POSPG chains. The POSPG/gelatin cogel has an elastomeric character with a very small value of loss tangent. The gelation kinetics and gel properties were discussed as a function of POSPG concentration, gelatin concentration, oxidation degree, temperature, and pH. This method can be used to design a large variety of cogels between SPG and proteins.     

Cloning of the transgenic pigs expressing human decay accelerating factor and N-acetylglucosaminyltransferase III

The present paper describes production of cloned pigs from fibroblast cells of transgenic pigs expressing human decay accelerating factor (DAF, CD55) and N-acetylglucosaminyltransferase III (GnT-III) that remodels sugar-chain biosynthesis. Two nuclear transfer protocols were used: a two-step activation (TA) method and a delayed activation (DA) method. Enucleated in vitro-matured oocytes and donor cells were electrically fused in a calcium-containing medium by TA method or in a calcium-free medium by DA method, followed by electrical activation 1-1.5 h later, respectively. In vitro blastocyst formation rates of nuclear transferred embryos reconstructed by TA and DA method were 8% and 14%, respectively. As a result of embryo transfer of the reconstructed embryos made by each method into recipient pigs, both gave rise to cloned piglets. These cloned pigs expressed transgene as much as their nuclear donor cells. In conclusions, (1) pig cloning can be carried out by TA or DA nuclear transfer methods, (2) expression of transgenes can be maintained to cloned pigs from the nuclear donor cells derived from transgenic animals.     

Fluorescent Immunochromatography for Rapid and Sensitive Typing of Seasonal Influenza Viruses

Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.