Selective expression of light-harvesting complexes alters phospholipid composition in the intracytoplasmic membrane and core complex of purple phototrophic bacteria.

Phospholipid-protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1-reaction center core complexes (LH1-RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1-RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex-phospholipid interactions.

Superconductivity induced by longitudinal ferromagnetic fluctuations in UCoGe.

From detailed angle-resolved NMR and Meissner measurements on a ferromagnetic (FM) superconductor UCoGe (T(Curie)∼2.5 K and T(SC)∼0.6 K), we show that superconductivity in UCoGe is tightly coupled with longitudinal FM spin fluctuations along the c axis. We found that magnetic fields along the c axis (H∥c) strongly suppress the FM fluctuations and that the superconductivity is observed in the limited magnetic-field region where the longitudinal FM spin fluctuations are active. These results, combined with model calculations, strongly suggest that the longitudinal FM spin fluctuations tuned by H∥c induce the unique spin-triplet superconductivity in UCoGe. This is the first clear example that FM fluctuations are intimately related with superconductivity.

Protective effect of ischaemic post-conditioning on ipsilateral and contralateral testes after unilateral testicular ischaemia-reperfusion injury.

The aim of this study was to investigate the effect of ischaemic post-conditioning (IPostC) against ischaemia-reperfusion (IR) injury on bilateral testes after unilateral testicular ischaemia in the rat. Eight-week-old male Sprague-Dawley rats were divided into control group; IR group (60 min ischaemia-24 h reperfusion); IPostC1 × 10 group (60 min ischaemia followed by one cycle of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion); IPostC3 × 10 group (three cycles of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion); IPostC5 × 10 group (five cycles of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion) and IPostC3 × 30 group (three cycles of 30 sec reperfusion-30 sec ischaemia; then 24 h reperfusion). In the IR and IPostC groups, the right testicular vessels were clamped using a special vascular clip. Malondialdehyde (MDA) and myeloperoxidase (MPO) levels were measured in testicular tissue samples bilaterally. Additionally, bilateral testicular tissue samples were processed for histological evaluation including haematoxylin-eosin, 4-hydroxy-2-nonenal (4-HNE) and TdT-mediated dUTP Nick End Labelling (TUNEL) staining. The levels of MDA and MPO as well as the positive cells per seminiferous tubule in TUNEL and 4-HNE stain in bilateral testes from the IR group were significantly higher compared with the control group. IPostC3 × 30 protocol significantly ameliorated the aforesaid parameters in both testes compared with the IR group. For the first time, we have demonstrated that IPostC protects both testes after unilateral testicular ischaemia-reperfusion. IPostC3 × 30 protocol offered the most effective protection.

Anisotropic magnetic fluctuations in the ferromagnetic superconductor UCoGe studied by direction-dependent 59Co NMR measurements.

We have carried out direction-dependent 59Co NMR experiments on a single crystal sample of the ferromagnetic superconductor UCoGe in order to study the magnetic properties in the normal state. The Knight-shift and nuclear spin-lattice relaxation rate measurements provide microscopic evidence that both static and dynamic susceptibilities are ferromagnetic with strong Ising anisotropy. We discuss that superconductivity induced by these magnetic fluctuations prefers spin-triplet pairing state.

Intra-articular distal ulnar fractures associated with distal radial fractures in older adults: early experience in fixation of the radius and leaving the ulna unfixed.

There is no clear consensus about the best management of intra-articular distal ulnar fractures associated with distal radial fractures in older adults. We describe a treatment wherein the distal radial fractures were securely fixed with a palmar plate, leaving the associated ulnar fractures unfixed. The wrists of 14 patients with a mean age of 74 years were reviewed at an average of 18 months after surgery. The results were excellent in 11 cases and good in three, according to the modified Gartland and Werley score. All fracture sites displayed union, and there was no instability of the distal radioulnar joint. A widening of the distal radioulnar joint space was present in one wrist. Angular deformity of the distal ulnar metaphysis was seen in five wrists. This treatment could be an alternative to open reduction with internal fixation for intra-articular distal ulnar fractures in older adults.

Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system.

An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines. The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of lambda(ex) = 415nm and lambda(em) = 485nm. A wide, linear relationship was obtained between the concentration of urea (1.0-100muM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.

Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats.

The effect of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of FITC-dextrans with a mean molecular weight ranging from 4.3 to 167 kDa and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats were studied. When FITC-dextrans were co-administered intranasally with 1.0 w/v% poly-L-Args of different molecular weight (MW, ca. 45.5 and 92 kDa, poly-L-Arg (50) and poly-L-Arg (100)), the bioavailability (F(infinity)) increased markedly compared with that after administration of FITC-dextran alone. However, the F(infinity) decreased exponentially with the increasing molecular weight of FITC-dextrans. There was no significant difference between the enhanced nasal absorption of FITC-dextrans achieved by the co-administration of poly-L-Arg (50) and poly-L-Arg (100). Moreover, the relationship between the F(infinity) and the molecular weight of FITC-dextrans indicated that the molecular weight of protein drugs, which exhibited efficient absorption with poly-L-Arg, was about 20 kDa, when the lower limit of bioavailability for developing a potent transnasal delivery system was assumed to be about 10%. Indeed, the nasal absorption of rhG-CSF, which has a molecular weight of 18.8 kDa, was also increased after co-administration of 1.0 w/v% poly-L-Arg (50) and the F(infinity) was about 11%. It seems likely that poly-L-Arg can be used to provide adequate nasal absorption of various protein drugs which have a molecular weight of about 20 kDa, thereby allowing the successful development of a variety of transnasal drug delivery systems.

Stem cell factor protects c-kit+ human primary erythroid cells from apoptosis.

OBJECTIVE: It has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC). Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit+ ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells. MATERIALS AND METHODS: Glycophorin A+ (GPA+) c-kit+ cells were purified from primary cultured ECFC derived from purified human CD34+ cells. The GPA+c-kit- and nonerythroid cells were generated from the same CD34+ cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry. For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting. RESULTS: In GPA+c-kit+ cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO. In contrast, no significant DNA fragmentation was seen in GPA+c-kit- and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF. PP2 also inhibited SCF-induced phosphorylation of Akt. CONCLUSION: These data indicate that SCF protects purified human GPA+c-kit+ cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival.

TY-12533, a novel Na(+)/H(+) exchange inhibitor, prevents myocardial stunning in dogs.

Anesthetized open-chest dogs were subjected to 15-min myocardial ischemia followed by 2-h reperfusion to induce myocardial stunning. A novel Na(+)/H(+) exchange inhibitor 6,7,8,9-tetrahydro-2-methyl-5H-cyclohepta[b]pyridine-3-carbonylguanidine maleate (TY-12533), administered 10 min before or 10 min after start of ischemia (3 mg/kg/10 min, i.v.), did not affect reductions in regional myocardial wall thickening, blood flow and pH during ischemia, but it significantly improved recovery of the wall thickening and blood flow after reperfusion. These results indicate that TY-12533, even when administered during ischemia, could prevent myocardial stunning without affecting myocardial dysfunction or acidosis induced by brief ischemia.

Possible participation of a JAK2 signaling pathway in recombinant rat interleukin-5-induced prolongation of rat eosinophil survival.

Recombinant rat interleukin (IL)-5-induced prolongation of rat eosinophil survival in culture was inhibited in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A when examined 96 h after incubation. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that the activation of JAK2 tyrosine kinase and protein synthesis are required for the prolongation of rat eosinophil survival induced by recombinant rat IL-5. STAT5 phosphorylation might also participate in the IL-5-induced survival of rat eosinophils.

Stem cell factor prevents Fas-mediated apoptosis of human erythroid precursor cells with Src-family kinase dependency.

The Fas ligand (Fas-L) expressed on mature erythroblasts may induce apoptosis of more immature erythroid cells that express Fas, whereas stem cell factor (SCF) may prevent Fas-mediated cell death in hematopoietic progenitor cells. The manner in which SCF prevents Fas-mediated cell death still is unclear. Given the essential role of SCF and the potentially important involvement of the Fas/Fas-L system in the development of erythrocytes, we studied mechanisms related to SCF prevention of Fas-mediated apoptosis. We used primary cultured human erythroid colony-forming cells (ECFC) derived from CD34+ cells and enriched glycophorin A positive (GPA+) c-kit+ cells in ECFC. Apoptosis of ECFC was induced by an Fas-L mimetic monoclonal antibody CH11. DNA fragmentation and the activation of caspase-3 and caspase-8 were measured using commercially available kits. Characterization of expanded cells was performed using multiparameter flow cytometry. Lyn kinase activity was measured by enolase kinase assays. SCF inhibited the CH11-induced DNA fragmentation of ECFC as well as enriched GPA+ c-kit+ cells in ECFC, but not those of GPA+ c-kit- cells. SCF also inhibited the activation of caspase-3 and caspase-8, without downregulation of the surface expression of Fas, suggesting that SCF prevents apoptosis through uncoupling of Fas ligation from subsequent caspase activation. PP2, a specific inhibitor of Src-family kinases, antagonized the effects of SCF in preventing Fas-mediated apoptosis. We propose that SCF prevents Fas-mediated apoptosis of erythroid progenitor cells in a manner dependent on the activity of Src-family tyrosine kinases. We also identified active Lyn in erythroid cells. These data suggest the presence of a novel Src-family-dependent function of SCF in the development of erythrocytes.

Grazing-exit X-ray spectrometry for surface and thin-film analyses.

Electron-probe x-ray micro analysis (EPMA) and particle-induced x-ray emission analysis (PIXE) were performed under grazing-exit conditions. To control the exit angle (take-off angle), a new sample holder having a stepping motor was developed for grazing-exit EPMA. A carefully polished surface of a stainless-steal sample was measured. The surface of stainless steel is normally covered with a thin native oxide layer. The intensity ratio of Cr K(alpha) to Fe K(alpha) increases significantly at the grazing angle, becoming about 5-times larger at 0.2 degrees than at 40 degrees. This result indicates that grazing-exit EPMA is useful for surface analysis. In addition, a new PIXE equipment was developed for grazing-exit x-ray measurements. The sample is fixed and the x-ray detector is moved by applying a linear stage. Preliminary experimental results of grazing-exit PIXE are also shown.

Analysis of the prolongation of rat eosinophil survival induced by recombinant rat interleukin-5.

Rat eosinophil survival was prolonged by recombinant rat IL-5 prepared by the baculovirus expression system. The IL-5-induced prolongation of eosinophil survival was dose-dependently inhibited by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that recombinant rat IL-5 activates JAK2 tyrosine kinase, which phosphorylates STAT5, and induces protein synthesis required for the prolongation of rat eosinophil survival.

Generation of rat eosinophils by recombinant rat interleukin-5 in vitro and in vivo.

The addition of recombinant rat interleukin-5 (IL-5), which was purified from the hemolymph of silkworm Bombyx mori larvae infected with IL-5-expressing recombinant virus, to cultures of rat bone marrow cells resulted in an increase in the number of Luxol-fast-blue staining eosinophils in a time- and concentration-dependent manner. After 6 days culture with 100 pM recombinant rat IL-5, more than 90% of the bone marrow cells were eosinophil. The contents of major basic protein (MBP) in the bone marrow cells determined by Western blot analysis using a polyclonal antibody to rat MBP were also increased by recombinant rat IL-5 (100 pM). Furthermore, intravenous injections of recombinant rat IL-5 twice a day for six consecutive days increased the population of eosinophils in peripheral blood cells and in bone marrow cells. These findings indicate that rat IL-5 induces terminal differentiation and proliferation of progenitor cells to mature eosinophils in rats.

Immunological and virological predictors of outcome during interferon-alpha therapy of chronic hepatitis C.

Results from a multicentre, clinical trial of interferon-alpha2a (IFN-alpha2a) for the treatment of chronic hepatitis C are reported. Serum hepatitis C virus (HCV) RNA levels were monitored as follows: before, and 2 days after, the first administration of IFN-alpha2a; during and at the end of treatment; and 6 months after completion of therapy. Peripheral blood lymphocyte subpopulations were measured, by two-colour flow cytometry, before and 3 h after the first intramuscular (i.m.) administration of 9 mega units (MU) of IFN-alpha2a. Virological responders had a significantly lower pretreatment level of CD11+ CD8- lymphocytes. Biochemical responders had significantly lower pretreatment levels of CD11- CD8+, human leucocyte antigen (HLA) DR- CD4- and HLA DR- CD8+ populations, and a higher pretreatment HLA DR+ CD4- population. These pretreatment differences disappeared 3 h after the first i.m. administration of IFN-alpha2a. CD11- CD8+ and HLA DR+ CD8+ cell populations became significantly lower in virological responders 3 h after the first i. m. administration of IFN-alpha2a. HLA DR+ CD4+ cell populations were increased less in biochemical responders. Thus, T-lymphocyte subpopulations were different between responders and non-responders to IFN therapy and IFN-modulated host immunity. Multivariate analysis showed that the pretreatment CD11+ CD8- cell population was an independent predictive factor of response to therapy. On the other hand, patients whose serum HCV RNA cleared or decreased within the first 2 days of IFN-alpha2a therapy were more likely to achieve a virological response. This predictive factor, however, was not an independent factor by multivariate analysis. These results suggest that host immunity is an important factor in response to IFN therapy, and HCV clearance within the first 2 days is a good predictive factor of response.

Sound-induced laryngeal and respiratory reflexes originate from vestibular afferents.

To determine whether the auditory or vestibular system causes the sound-induced laryngeal reflex, which has been considered to participate in the auditory feedback control of vocalization, click-induced laryngeal responses were compared before and after sectioning of the cochlear and/or vestibular nerves in cats. The sound-induced reflex modulation of respiratory muscle activity was also investigated, because respiratory movement is important for vocal control. Sectioning of the cochlear nerves had little influence on these responses. In contrast, sectioning of the vestibular nerves abolished these responses. It was concluded that the sound-induced laryngeal and respiratory reflexes are attributed to the vestibular system.

Analysis of biological activities of recombinant rat interleukin-5.

We analyzed the biological activities of recombinant rat interleukin-5 (IL-5). Purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5-neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in eosinophil viability was inhibited in a time- and concentration-dependent manner. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5-neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B cell growth factor II, eosinophil differentiation factor, and eosinophil survival-enhancing factor.

Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities.

Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor.

Biosorption of actinides from dilute waste actinide solution by egg-shell membrane.

Removal of radioactive elements from the effluent and waste aqueous solutions is an important problem. In previous laboratory batch experiments, hen egg-shell membrane (ESM) was stable as an insoluble protein and was very capable of binding heavy metal ions from aqueous solution. Batch laboratory pH profile, time dependency, and capacity experiments were performed to determine the binding of uranium (U) and thorium (Th) to ESM. Batch pH profile experiments indicated that the optimum pH for binding these actinides was approx 6.0 (U) or 3.0 (Th). The adsorption isotherms were developed at pH 5.0 (U) or 3.0 (Th) at 25 degrees C, and the adsorption equilibrium data fitted both Langmuir and Freundlich models. The maximum uptakes by the Langmuir model were about 240 mg U/g and 60 mg Th/g dry weight ESM. In addition, their adsorption capacities increased as salt concentration increased. ESM could also accumulate uranium from dilute aqueous solution by adjusting to the optimum pH. These results showed that ESM was effective for removing actinides from solution and would be useful in filtration technology to remove actinides from aqueous solution.

Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis.

The nucleotide sequences of the 16S rRNA gene (rDNA) in 38 taxa of the genus Staphylococcus were compared phylogenetically. Based on phylogenetic tree analysis, staphylococcal species were divided into 12 cluster groups. These cluster groups were in very good agreement with species groups determined by DNA-DNA reassociation studies. These genealogical classifications were consistent with the results of the production of coagulase or oxidase and with resistance to novobiocin. These suggest that the phylogenetic relationship of the genus Staphylococcus is accurately represented by the results obtained from the sequence analysis of 16S rDNA.