Biomedical subjects

Takashi Fukui

Publications and source records attributed to Takashi Fukui.

9 recordsLinked to original sources

PubMed2006

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy.

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 A. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 A. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C(4) symmetric particles with edge and diagonal lengths of 65 A and 80 A, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C(4) symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.

Bacterial Toxins↗

PubMed2006

Differences in protein synthesis between wild type and intracellular growth-deficient strains of Legionella pneumophila in U937 and Acanthamoeba polyphaga.

An important aspect of Legionnaires' disease is the growth of the causative agent, Legionella pneumophila, within infected host cells. Many proteins including stress proteins of L. pneumophila were strongly induced in a wild type strain that had been used to infect U937 human macrophage-like cells. In contrast, the expression of the proteins was much weaker within a protozoan host, Acanthamoeba polyphaga. The results suggested that active bacterial protein synthesis is required more within macrophages than within protozoa for adaptation of L. pneumophila to intracellular environments. The synthesis of these proteins was not observed in intracellular growth-deficient strains after infection in either type of host cells. The inability of protein synthesis in these strains is correlated with their inability of intracellular growth. Furthermore, on U937 infection, the synthesis of beta-galactosidase encoded in an inducible reporter construct immediately ceased in the in intracellular growth-deficient strains after infection, while the wild type strain was able to synthesize it during the course of infection. These results suggested that the intracellular growth of Legionella pneumophila within macrophages requires active protein synthesis from an earlier stage of bacterial infection.

Acanthamoeba↗

PubMed2005

Thermostable direct hemolysin of Vibrio parahaemolyticus is a bacterial reversible amyloid toxin.

Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, is detoxified by heating at approximately 60-70 degrees C but is reactivated by additional heating above 80 degrees C. This paradoxical phenomenon, known as the Arrhenius effect, has remained unexplained for approximately 100 years. We now demonstrate that the effect is related to structural changes in the protein that produce fibrils. The native TDH (TDHn) is transformed into nontoxic fibrils rich in beta-strands by incubation at 60 degrees C (TDHi). The TDHi fibrils are dissociated into unfolded states by further heating above 80 degrees C (TDHu). Rapid cooling of TDHu results in refolding of the protein into toxic TDHn, whereas the protein is trapped in the TDHi structure by slow cooling of TDHu. Transmission electron microscopy indicates the fibrillar structures of TDHi. The fibrils show both the property of the nucleation-dependent elongation and the increase in its thioflavin T fluorescence. Formation of beta-rich structures of TDH was also observed in the presence of lipid vesicles containing ganglioside G(T1b), a putative TDH receptor. Congo red was found to inhibit the hemolytic activity of TDH in a dose-dependent manner. These data reveal that the mechanism of the Arrhenius effect which is tightly related to the fibrillogenicity of TDH.

Amyloid↗

PubMed2005

A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system.

pH-Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluorescence, and sedimentation equilibrium ultracentrifugation. Far- and near-UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity (in the presence of EspA) was observed at acidic pH; whereas, no increased ANS fluorescence was observed at pH 7.0. These results suggest the presence of a partially unfolded state. Interestingly, urea-induced unfolding transitions, monitored by far-UV CD spectroscopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH. Although increased ANS fluorescence was observed at pH 3.0, the urea-induced unfolding curve is similar to that found at pH 7.0. This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule. The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing alpha-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular beta-sheets at pH 2.0. Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH.

Amino Acid Sequence↗

PubMed2003

Localized relapse in bone marrow in a posttransplantation patient with t(6;9) acute myeloid leukemia.

We report a 38-year-old woman with t(6;9) acute myeloid leukemia who relapsed with localized leukemic cell growth in the bone marrow after she had undergone allogeneic bone marrow transplantation. The localized cell growth was first recognized by an apparent discrepancy in the DEK-CAN fusion transcript levels between the aspirates from the left and right iliac bone marrow. Magnetic resonance imaging of the iliac bone revealed localized cell accumulation in the left side. The nonhomogeneous and localized leukemic cell growth in this case may have been due to the graft-versus-leukemia effect following allogeneic transplantation with donor lymphocyte infusion. Serial monitoring of molecular markers for leukemia at different sites or magnetic resonance imaging of the bone marrow may be of value in detecting this type of relapse.

Adult↗

PubMed2003

Restricted expression of shiga toxin binding sites on mucosal epithelium of mouse distal colon.

Shiga toxins (Stx) are some of the major virulence factors of enterohemorrhagic Escherichia coli strains such as serotype O157:H7. To explore how Stx might initially gain access to the bloodstream from sites of infection, frozen sections of mouse colon were immunohistochemically examined for binding sites for recombinant binding subunits (Stx1B). Binding sites were selectively expressed on the epithelium in the distal half of the mouse colon, whereas the proximal half did not exhibit any binding sites. In agreement with this observation, we also demonstrated the distal-part-restricted distribution of glycolipids that bind to Stx1B in the mouse colon. For comparison, the binding sites of several control lectins were also examined. Selective binding to the distal part of the colon was not seen with any other control lectins, including Griffonia simplicifolia lectin-I isolectin B4 (GS-I-B4), which shares alpha-galactose specificity with Stx1B. Partial overlapping of the specificities of Stx1B and GS-I-B4 was seen by assay with globotriose-conjugated multivalent ligands. The results indicate that Stx1B is stricter in the recognition of carbohydrate determinants than GS-I-B4 when examined with biological ligands.

Animals↗

PubMed2002

Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues.

B cells in germinal centres are known to express carbohydrate antigen CD77 in human lymphoid tissues. The CD77 antigen is specifically recognized by Shiga-like toxins (SLTs) that are produced by enterohaemorrhagic Escherichia coli O157:H7. To determine whether the binding subunits of Shiga-like toxin-1 (SLT-1B) could have adverse effects on the murine immune system when used as an immunogen, we investigated whether SLT-1B could bind to germinal centres of mouse lymphoid tissues. Frozen sections of peripheral lymph nodes and Peyer's patches from immunized mice were tested for the presence of SLT-1B-binding sites by immunohistological methods. Germinal centres were not stained with SLT-1B, while they were intensely stained with peanut agglutinin (PNA), another marker of germinal centres. On the other hand, SLT-1B specifically bound to renal tubules and collecting ducts in frozen sections of mouse kidney. This is consistent with results from human tissues. We also demonstrated that B220/PNA double-positive populations in lymph nodes from immunized mice exhibited only marginal staining with SLT-1B. The present results suggest that SLTs would not impede germinal centre functions of the murine immune system.

Animals↗

PubMed2002

Quantitative measurement of 17 beta-estradiol and estriol in river water by time-resolved fluoroimmunoassay.

A sensitive method for detecting 17 beta-estradiol (E2) and estriol (E3) in river water has been developed, based on the time-resolved fluoroimmunoassay by using a fluorescent europium chelate label, 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl)- chlorosulfo-o-terphenyl (BHHCT)-Eu3+. In the E2 assay, microtiter plates were coated with the E2-bovine serum albumin (BSA) conjugate. The anti-17 beta-estradiol antibody, the biotinylated goat anti-rabbit IgG antibody and the BHHCT-Eu3+ labeled streptavidin (SA)-BSA conjugate were used. In the E3 assay, the goat anti-rabbit IgG antibody was coated on a microtiter plate. The anti-estriol antibody and the BHHCT-Eu3+ labeled E3-BSA conjugate were used. The detection limits for E2 and E3 were 2.3 pg/ml and 4.3 pg/ml, respectively, and the analytical recoveries were 95-120%. Quantitative measurement of estrogens in river water was carried out for Kanda River (Tokyo, Japan) by using the method. The E2 and E3 levels were 32 pg/ml and 5.5 pg/ml, respectively. The detection limits of the present method are in the same orders of magnitude as those of ELISA for E2, and are 1-2 orders of magnitude better for E3.

Animals↗

PubMedDate not supplied

A majority-logic nanodevice using a balanced pair of single-electron boxes.

This paper describes a majority-logic gate device that will be useful in developing single-electron integrated circuits. The gate device consists of two identical single-electron boxes combined to form a balanced pair. It accepts three inputs and produces a majority-logic output by using imbalances caused by the input signals; it produces a 1 output if two or three inputs are 1, and a 0 output if two or three inputs are 0. We combine these gate devices into two subsystems, a shift register and an adder, and demonstrate their operation by computer simulation. We also propose a method of fabricating the unit element of the gate device, a minute dot with four coupling arms. We demonstrate by experiments that it is possible to arrange these unit elements on a GaAs substrate, in a self-organizing manner, by means of a process technology that is based on selective-area metalorganic vapor-phase epitaxy.

Aluminum↗