Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.     

Isoprostanes, prostaglandins and tocopherols in pre-eclampsia, normal pregnancy and non-pregnancy

This study is designed to evaluate whether oxidative stress and inflammation are involved in severe pre-eclampsia compared to normal pregnancy and non-pregnancy. We have measured plasma and urinary levels of 8-iso-PGF2alpha, a major isoprostane as an indicator of oxidative stress; plasma and urinary 15-keto-dihydro-PGF2alpha, a major metabolite of cyclooxygenase-catalysed PGF2alpha as an indicator of inflammatory response, and plasma -alpha-and -gamma-tocopherol in 18 pre-eclamptic, 19 normal pregnancy and 20 non-pregnant women. Pregnant women had significantly higher levels of 8-iso-PGF2alpha and PGF2alpha metabolite as compared to the non-pregnancy. Levels of 8-iso-PGF2alpha in the pre-eclamptic women did not differ from the normal pregnancy but PGF2alpha metabolite levels were significantly higher in normal pregnancy. On the other hand, gamma-tocopherol levels were significantly lower in pre-eclampsia than normal pregnancy. In contrast, the concentration of alpha-tocopherol was very similar between the groups. alpha-and gamma-tocopherol levels were significantly lower in pregnancy compared to non-pregnancy. Although no direct evidence of oxidative stress and inflammatory response was observed in severe pre-eclampsia, a reduction of gamma-tocopherol suggests the possible precedence of oxidative stress in this condition. Higher levels of isoprostanes and prostaglandin metabolite in late pregnancy suggest the importance of both free radicals and cyclooxygenase-catalysed oxidation products in normal biological processes of pregnancy.     

Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells

Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression.     

<Emphasis Type="Italic">Sporotomaculum syntrophicum</Emphasis> sp. nov., a novel anaerobic,syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).     

In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts

Time-sequential responses to endothelium-dependent and -independent vasodilators and angiotensin-converting enzyme (ACE) inhibitors were studied in the subendocardial arterioles (Endo) of canine renovascular hypertension (HT) compared with subepicardial arterioles (Epi; both <120 microm) by charge-coupled device intravital microscope. Vascular responses to acetylcholine, papaverine, and cilazaprilat were compared between normotensive (NT) and HT dogs [4 wk and 12 wk of HT (4wHT and 12wHT)]. The acetylcholine-induced vasodilation of Endo in both 4wHT and 12wHT was smaller than that of NT (both P < 0.01 vs. 4wHT and 12wHT), and that of Epi was smaller than that of NT only in 12wHT (P < 0.05). The papaverine-induced vasodilation of Endo, but not Epi, was impaired only in 12wHT (both P < 0.01 vs. NT and 4wHT). Vasodilation by cilazaprilat remained unchanged at 4wHT and 12wHT in both Epi and Endo. In conclusion, at the early stage, the endothelium-dependent response of Endo was impaired, whereas at the later stage, the endothelium-dependent and -independent responses of Endo and the endothelium-dependent response of Epi were impaired. However, the vasodilatory responses to the ACE inhibitor were maintained in both Endo and Epi of HT.     

Intramuscular gene transfer of FGF-2 attenuates endothelial dysfunction and inhibits intimal hyperplasia of vein grafts in poor-runoff limbs of rabbit

We previously demonstrated that sustained disturbance of endothelium-dependent vasorelaxation and poor distal runoff in ischemic limbs were critical factors affecting the neointimal development of autologous vein grafts (VGs). Also, we recently showed the superior therapeutic potential of basic fibroblast growth factor (bFGF/FGF-2) boosted by the recombinant Sendai virus (SeV) for severe limb ischemia compared with that of vascular endothelial growth factor. Here, the effect of FGF-2 on neointimal hyperplasia of VGs was examined in a rabbit model of poor-runoff limbs. Two weeks after initial surgery for the induction of poor-runoff, SeV-expressing human FGF-2 (SeV-hFGF2) or that encoding firefly luciferase (109 plaque-forming units/head) was injected into the thigh and calf muscle. At that time, the femoral vein was implanted in the femoral artery in an end-to-end manner in some groups. FGF-2 gene-transferred limbs demonstrated significantly increased blood flow assessed not only by laser Doppler flow image but also by ultrasonic transit-time flowmeter (USTF). USTF also showed a significant increase in the blood flow ratio of the deep femoral artery to external iliac artery, indicating that collateral flow was significantly restored in the thigh muscles (P < 0.01). Reduction of neointimal hyperplasia was also observed in the VGs treated by SeV-hFGF2; these grafts demonstrated significant restoration of endothelium-dependent vasorelaxation. These findings thus extend the indications of therapeutic angiogenesis using SeV-hFGF2 to include not only limb salvage but also prevention of late graft failure.     

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin

Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.     

Torque-speed relationship of the Na+-driven flagellar motor of Vibrio alginolyticus

The torque-speed relationship of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated. The rotation rate of the motor was measured by following the position of a bead, attached to a flagellar filament, using optical nanometry. In the presence of 50mM NaCl, the generated torque was relatively constant ( approximately 3800pNnm) at lower speeds (speeds up to approximately 300Hz) and then decreased steeply, similar to the H(+)-driven flagellar motor of Escherichia coli. When the external NaCl concentration was varied, the generated torque of the flagellar motor was changed over a wide range of speeds. This result could be reproduced using a simple kinetic model, which takes into consideration the association and dissociation of Na(+) onto the motor. These results imply that for a complete understanding of the mechanism of flagellar rotation it is essential to consider both the electrochemical gradient and the absolute concentration of the coupling ion.