Reactivities of the coordinated organonitriles in molybdenum(0) and tungsten(0) phosphine complexes: protonation of the nitrile carbon and cleavage of the CN triple bond

The molybdenum and tungsten dinitrogen-organonitrile complexes trans-[M(N₂)(NCR)(dppe)₂] (2, M=Mo; 4, M=W; R=Ph, C₆H₄Me-p, C₆H₄OMe-p, Me; dppe=Ph₂PCH₂CH₂PPh₂) underwent double protonation at the nitrile carbon atom with loss of N₂ and a change in oxidation state to +4 on treatment with hydrochloric acid to afford the cationic imido complexes trans-[MCl(NCH₂R)(dppe)₂]⁺. The solid-state structure of trans-[WCl(NCH₂CH₃)(dppe)₂][PF₆]·CH₂Cl₂ was determined by single-crystal X-ray analysis. Protonation of complexes 2 by fluoroboric acid or hydrobromic acid also formed the similar imido complexes trans-[MoX(NCH₂R)(dppe)₂]⁺ (X=F, Br). In contrast, the dinitrogen complex trans-[Mo(N₂)₂(dppe)₂] reacted with two equiv. of benzoylacetonitrile, a nitrile with acidic CH hydrogen atoms, to give the nitrido complex trans-[Mo(N)(NKCCHCOPh)(dppe)₂] (12), which was accompanied by evolution of dinitrogen and the formation of 1-phenyl-2-propen-1-one in high yields. For complex 12, the zwitterionic structure, where the anionic enolate ligand PhC(O⁺)=CHCN coordinates to the cationic Mo(IV) center through its nitrogen atom, was confirmed by spectroscopic measurements and single-crystal X-ray analysis. A unique intermolecular aromatic C---HO hydrogen bonding was observed in that crystal structure. Complex 12 is considered to be formed via the cleavage of the CN triple bond of benzoylacetonitrile on the metal. A reaction mechanism is proposed, which includes the double protonation of the nitrile carbon atom of the ligating benzoylacetonitrile on a low-valent molybdenum center.     

Knock Out of S1P3 Receptor Signaling Attenuates Inflammation and Fibrosis in Bleomycin-Induced Lung Injury Mice Model

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.     

Rapid detection of quinolone-resistant Salmonella by real time SNP genotyping

A total of 63 isolates were screened for the gyrA mutation (87Asp-Tyr) in Salmonella enterica serovars using real time PCR. All of the isolates were successfully identified as resistant or susceptible, consistent with the MIC result of the agar dilution method and gyrA sequencing.     

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-D-erythro-sphingosine (C16Cer), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (C16SM) and cholesterol. The amounts of DOPC, sum of C16Cer and C16SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C16Cer/(C16Cer+C16SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C16Cer (X=0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X=0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X>or=0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: changes in nutrient concentrations,phytoplankton and zoobenthos

Large bag-type (75 m³) and tube-type (105 m³) enclosures were set up in the shallow eutrophic Lake Suwa and were each stocked with exotic planktivorous whitefish (Coregonus lavaretus maraena). The release of whitefish caused the increase in nutrient concentration in the tube-type enclosure whereas no such increase was observed in the bag-type enclosure. Bottom sediment seemed to be an important source of chironomid food for whitefish. The proportion of phytoplankton measuring<10μm and 20–40μm, which respectively corresponded toOchromonas spp. andCryptomonas sp., were lower in the fish enclosures than in the control, which might have been caused by high grazing pressure by rotifers. The predation by whitefish might have affected the species composition of phytoplankton through reducing copepod predation on rotifers, not through reducing the densities of cladocerans which directly feed on phytoplankton as many investigators have reported. The phytoplankton biomass was not affected much by the release of fish. Possible reasons are that the increase in density of rotifers reduced the biomass of available phytoplankton and also that inedible Cyanophyceae were in the decreasing phase of their seasonal succession and could not increase successfully in spite of elevated nutrient levels.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

Involvement of regulatory T cells in the experimental autoimmune encephalomyelitis-preventive effect of dendritic cells expressing myelin oligodendrocyte glycoprotein plus TRAIL

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4(+)CD25(+) regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4(+)CD25(+) T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3(+) cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4(+)CD25(+) T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4(+)CD25(+) Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.     

Actin filament organization of foot processes in rat podocytes.

The foot processes of podocytes possess abundant microfilaments and modulate glomerular filtration. We investigated the actin filament organization of foot processes in adult rat podocytes and the formation of the actin cytoskeletal system of immature podocytes during glomerulogenesis. Electron microscopy revealed two populations of actin cytoskeletons in foot processes of adult podocytes. One is the actin bundle running above the level of slit diaphragms and the other is the cortical actin network located beneath the plasmalemma. Immunogold labeling for actin-binding proteins demonstrated that alpha-actinin and synaptopodin were localized in the actin bundle, whereas cortactin was in the cortical actin network. Immunofluorescence labeling for actin-binding proteins in immature podocyte showed that alpha-actinin was localized at the level of the junctional complex, whereas cortactin was distributed beneath the entire plasmalemma. Synaptopodin was first observed along the basal plasmalemma from the advanced S-shaped body to the capillary loop stage. We conclude that foot processes have specialized actin filamentous organization and that its establishment is associated with the expression and redistribution of actin-binding proteins during development.     

Glucose 1-phosphate increases active transport of calcium in intestine

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.     

Impact of whitefish on an enclosure ecosystem in a shallow eutrophic lake: selective feeding of fish and predation effects on the zooplankton communities

Bag-type enclosures (75 m³) with bottom sheets and tube-type enclosures (105 m³) open to the bottom sediment were stocked with exotic whitefish (Coregonus lavaretus maraena) to study their predation effects on the plankton community. The fish fed mainly on adult chironomids during the period of their emergence (earlier part of the experimental period). Thereafter, the food preference was shifted to larvae of chironomids and crustacean zooplankters. The predation effects on the plankton community were not evident in the bag-type enclosures where zooplankton densities were consistently low. The fish reduced the crustacean populations composed ofBosmina fatalis, B. longirostris andCyclops vicinus in the tube-type enclosures where the prey density was high (above ca. 50 individuals 1⁻¹). The results suggested that the intensity of predation depended on the prey density. Rotifers increased in the fish enclosure, probably becauseCoregonus reduced the predation pressure byCyclops vicinus on rotifers and allowed the latter to increase. In the fish enclosures, no marked changes in species composition were observed. Zooplankton predated by the fish seemed to be distributed near the walls of the enclosures. Problems of enclosure experiments for examining the effects of fish predation on pelagic zooplankton communities are discussed.