Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.     

Small angle X-ray scattering studies on local structure of tobacco mosaic virus RNA in solution

Effects of temperature and ionic strength (S) on the local structure of tobacco mosaic virus RNA in phosphate buffer solution are studied by analyzing the small-angle X-ray scattering (SAXS) curves. The root-mean-square radius of a cross-section of RNA chain was kept at 0.845+/-0.005 nm over a wide range of S from 0.2 to 0.003 at 20 degrees C, whereas it gradually diminished from 0.85 to 0.61 nm when the temperature is raised from 20 to 50 degrees C at S = 0.2. Nevertheless, all of SAXS curves reflecting the backbone structures were equally mimicked by theoretical ones of freely hinged rod (FHR) models, i.e. several straight rods joined with freely hinged joints in the form of a combination of the letter Y, if the constituent rod lengths in the models are adjusted. From these facts, it is suggested that the local structure of the RNA chain in aqueous solution is characterized by an essential feature that unpaired bases in the partially double-stranded helix are constantly far isolated from each other along the helix and the rod-like structure of the helix is preserved over a range of helical contents. Such a characteristic local structure of the chain is entirely collapsed in the formamide solution at 50 degrees C.     

Analysis of human abnormal walking using a multi-body model: joint models for abnormal walking and walking aids to reduce compensatory action

This paper proposes new models of diseased joints and evaluates the effectiveness of walking aids such as a cane and a brace for compensating for lost functions due to joint disorders. The ZMJ concept described in the previous work (Yamashita and Tagawa, 1988. In: Radharaman (Ed.), Robotics and Factories of the Future'87. Springer, New York, pp. 670-677) is modified into three joint models as follows: a passive element joint (PEJ) which has a spring at the diseased joint; a constrained range joint (CRJ), the motion of which stays within some constrained relative angle; a partial moment joint (PMJ) which can produce a partial amount of the moment produced about the joint in normal walking. A cane can enlarge a supporting area and adjust the posture of the upper torso to be upright. An ischial weight-bearing brace is effective for conservative management of hip disorders by reducing a load to the joint (Shiba et al., 1998. Clinical Orthopaedics and Related Research 351, 149-157). Walking aids like a cane or brace have been conveniently used by the handicapped. Abnormal walking was simulated for each joint model. Dynamic effects of a cane and a brace on abnormal walking were examined by the multi-body walking model.     

Interferon gamma priming is not critical for IL-12 production of murine spleen cells

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.     

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2′-deoxyuridine

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology.     

Transient formation of a neutral ubisemiquinone radical and subsequent intramolecular electron transfer to pyrroloquinoline quinone in the Escherichia coli membrane-integrated glucose dehydrogenase

The membrane-bound quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli contains pyrroloquinoline quinone (PQQ) and participates in the direct oxidation of D-glucose to D-gluconate by transferring electrons to ubiquinone (UQ). To elucidate the mechanism of ubiquinone reduction by mGDH, we applied a pulse radiolysis technique to mGDH with or without bound UQ8. With the UQ8-bound enzyme, a hydrated electron reacted with mGDH to form a transient species with an absorption maximum at 420 nm, characteristic of formation of a neutral ubisemiquinone radical. Subsequently, the decay of the absorbance at 420 nm was accompanied by an increase in the absorbance at 370 nm. Experiments with the PQQ-free apoenzyme showed no such subsequent absorption changes, although ubisemiquinone was formed. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone radical at the UQ8 binding site to PQQ exists in mGDH. The first-order rate constant of this process was calculated to be equal to 1.2 x 10(3) s(-1). These findings are consistent with our proposal that during the catalytic cycle of mGDH the bound UQ8 mediates electron transfer from the reduced PQQ to UQ8 pools.     

The role of gamma interferon in acquired host resistance against Staphylococcus aureus infection in mice

We investigated the expression of an acquired host resistance against Staphylococcus aureus infection in mice. When C57BL/6 mice were immunized with viable S. aureus and challenged with S. aureus eight weeks later, the elimination of S. aureus from the spleen and liver was enhanced in the immunized mice compared with the nonimmunized mice. When gamma interferon (IFN-gamma(-/-)) mice were immunized and challenged, the bacterial numbers in the organs of immunized mice were comparable to those in the nonimmunized mice, suggesting that IFN-gamma plays a critical role in an acquired host resistance against S. aureus infection. IFN-gamma(-/-) mice produced the lower level of anti-S. aureus immunoglobulin M (IgM) and IgG2a antibodies compared with C57BL/6 mice. To elucidate the role of IFN-gamma produced during a challenge with S. aureus, a single injection of anti-IFN-gamma monoclonal antibody to mice was carried out 1 h before challenge. An acquired resistance against S. aureus infection was inhibited by injecting with anti-IFN-gamma monoclonal antibody. However, anti-IFN-gamma monoclonal antibody treatment failed to modulate anti-S. aureus IgM, IgG1 or IgG2a responses in these animals. These results demonstrated that IFN-gamma is required for an acquired resistance against S. aureus infection in mice. However, IFN-gamma induced during the challenge failed to affect the secondary antibody responses.