Role of interaction of mannan-binding protein with meprins at the initial step of complement activation in ischemia/reperfusion injury to mouse kidney

Ischemia/reperfusion (I/R) is an important cause of acute renal failure. Recent studies have shown that the complement system mediated by the mannan-binding protein (MBP), which is a C-type serum lectin recognizing mannose, fucose and N-acetylglucosamine residues, plays a critical role in the pathogenesis of ischemic acute renal failure. MBP causes complement activation through the MBP lectin pathway and a resulting complement component, C3b, is accumulated on the brush borders of kidney proximal tubules in a renal I/R-operated mouse kidney. However, the initial step of the complement activation has not been studied extensively. We previously identified both meprins α and β, highly glycosylated zinc metalloproteases, localized on kidney proximal tubules as endogenous MBP ligands. In the present study, we demonstrated that serum-type MBP (S-MBP) and C3b were co-localized with meprins on both the cortex and the medulla in the renal I/R-operated mouse kidney. S-MBP was indicated to interact with meprins in vivo in the I/R-operated mouse kidney and was shown to initiate the complement activation through the interaction with meprins in vitro. Taken together, the present study strongly suggested that the binding of S-MBP to meprins triggers the complement activation through the lectin pathway and may cause the acute renal failure due to I/R on kidney transplantation and hemorrhagic shock.     

Efficient iPS cell production with the MyoD transactivation domain in serum-free culture

A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.     

Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells

Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.     

Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.)

Powdery mildew caused by Podosphaera xanthii is an important foliar disease in melon. To find molecular markers for marker-assisted selection, we constructed a genetic linkage map of melon based on a population of 93 recombinant inbred lines derived from crosses between highly resistant AR 5 and susceptible ‘Earl’s Favourite (Harukei 3)’. The map spans 877 cM and consists of 167 markers, comprising 157 simple sequence repeats (SSRs), 7 sequence characterized amplified region/cleavage amplified polymorphic sequence markers and 3 phenotypic markers segregating into 20 linkage groups. Among them, 37 SSRs and 6 other markers were common to previous maps. Quantitative trait locus (QTL) analysis identified two loci for resistance to powdery mildew. The effects of these QTLs varied depending on strain and plant stage. The percentage of phenotypic variance explained for resistance to the pxA strain was similar between QTLs (R ² = 22–28%). For resistance to pxB strain, the QTL on linkage group (LG) XII was responsible for much more of the variance (41–46%) than that on LG IIA (12–13%). The QTL on LG IIA was located between two SSR markers. Using an independent population, we demonstrated the effectiveness of these markers. This is the first report of universal and effective markers linked to a gene for powdery mildew resistance in melon.     

Chemical anatomy of hydra nervous system using antibodies against hydra neuropeptides: a review

Polyclonal antibodies against hydra neuropeptides allow us to visualize the hydra nerve net. Chemical anatomy of the hydra nerve net was archived by means of immunocytochemistry with various antibodies to hydra neuropeptides. Our goal is to describe the hydra nerve net both qualitatively and quantitatively. The present chemical anatomy results indicate (1) differences in peptide expression between ganglion cells and sensory cells, (2) large differences in neuropeptide expression between ectodermal nerve cells and endodermal nerve cells, and (3) the usefulness of quantitative chemical anatomy to analyze the entire nervous system of hydra.     

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Isolation of stable (alphabeta)4-Tetraprotomer from Na+/K+-ATPase solubilized in the presence of short-chain fatty acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) > Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.     

Does Waist Circumference Add to the Predictive Power of the Body Mass Index for Coronary Risk?

Objectives: To examine the power of the combined measurements of body mass index (BMI) and waist circumference (WC) for the prediction of abnormality in coronary heart disease risk factors and to determine whether the additional measurement of WC is predictive in older men and women. Research Methods and Procedures: 1190 men and 751 women of the Baltimore Longitudinal Study of Aging were dichotomized into younger (<65 years) and older (65+ years) age groups. Coronary risk factors in the realms of glucose/insulin metabolism, blood pressure, and plasma lipids were assessed. The relationship of BMI and WC, singly and combined, to 10 risk factors for coronary heart disease was examined. Results: In younger and older men and women, BMI and WC are highly correlated (0.84 to 0.88). BMI and WC are also significantly correlated to all 10 coronary risk factors in younger men and women and to 8 of the 10 in the older men and women. Both partial correlation and logistic regression analyses revealed a modest but significant improvement in the prediction of coronary risk in younger men and women by WC after controlling for the level of BMI. There was no improvement in the older subjects. Discussion: WC adds only modestly to the prediction of coronary risk in younger subjects once BMI is known, and adds nothing to the production of risk in older subjects.     

Adaptive response of Schizosaccharomyces pombe to hydrogen peroxide

Abstract Schizosaccharomyces pombe becomes resistant to killing by high concentration of hydrogen peroxide and other severe stresses including oxidants, high temperature and high concentration of ethanol when pretreated with nonlethal levels of hydrogen peroxide. In the presence of the protein synthesis inhibitor, cycloheximide, during hydrogen peroxide pretreatment, the cell obtained partial resistance to a higher level of hydrogen peroxide. The partial resistance to hydrogen peroxide in the presence of cycloheximide was acquired within 30 min of pretreatment but complete resistance obtained with de novo protein synthesis was not attained before 45 min of pretreatment. During adaptation to hydrogen peroxide, at least 15 polypeptides are induced, as analyzed by two-dimensional gel electrophoresis. Catalase activity is induced eight-fold by treatment with a nonlethal level of hydrogen peroxide.