Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer’s patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.     

Metabolism of 3,4-Dimethylphenyl N-Methylcarbamate in White Rats

Orally administered 3,4-dimethylphenyl N-methylcarbamate labelled with carbon-14 at 4-CH₃ was easily absorbed from the gastrointestinal tract of male Wistar rats and distributed into the tissues. Elimination of the radioactivity was rapid and essentially complete; namely during 48 hr approximately 92% and 5% of the total radioactivity were excreted respectively into urine and feces. The content of the intact carbamate compound in the urine was less than 0.5%. Major degradation products were identified as 3-methyl-4-carboxyphenyl N-methylcarbamate, its N-hydroxymethyl analog and its component phenol. Much less amount of direct hydrolysis product of the original carbamate, 3,4-dimethylphenol and its conjugated forms was demonstrated. 3,4-Dimethylphenyl N-methylcarbamate is presumed to undergo biodegradation through oxidative pathways.     

Potential Measurement Errors Due to Image Enlargement in Optical Coherence Tomography Imaging

The effect of interpolation and super-resolution (SR) algorithms on quantitative and qualitative assessments of enlarged optical coherence tomography (OCT) images was investigated in this report. Spectral-domain OCT images from 30 eyes in 30 consecutive patients with diabetic macular edema (DME) and 20 healthy eyes in 20 consecutive volunteers were analyzed. Original image (OR) resolution was reduced by a factor of four. Images were then magnified by a factor of four with and without application of one of the following algorithms: bilinear (BL), bicubic (BC), Lanczos3 (LA), and SR. Differences in peak signal-to-noise ratio (PSNR), retinal nerve fiber layer (RNFL) thickness, photoreceptor layer status, and parallelism (reflects the complexity of photoreceptor layer alterations) were analyzed in each image type. The order of PSNRs from highest to lowest was SR > LA > BC > BL > non-processed enlarged images (NONE). The PSNR was statistically different in all groups. The NONE, BC, and LA images resulted in significantly thicker RNFL measurements than the OR image. In eyes with DME, the photoreceptor layer, which was hardly identifiable in NONE images, became detectable with algorithm application. However, OCT photoreceptor parameters were still assessed as more undetectable than in OR images. Parallelism was not statistically different in OR and NONE images, but other image groups had significantly higher parallelism than OR images. Our results indicated that interpolation and SR algorithms increased OCT image resolution. However, qualitative and quantitative assessments were influenced by algorithm use. Additionally, each algorithm affected the assessments differently.     

Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.     

DNase γ Is the Effector Endonuclease for Internucleosomal DNA Fragmentation in Necrosis

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg²⁺/Ca²⁺-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Genome-Wide Association Study of Serum Creatinine Levels during Vancomycin Therapy

Vancomycin, a commonly used antibiotic, can be nephrotoxic. Known risk factors such as age, creatinine clearance, vancomycin dose / dosing interval, and concurrent nephrotoxic medications fail to accurately predict nephrotoxicity. To identify potential genomic risk factors, we performed a genome-wide association study (GWAS) of serum creatinine levels while on vancomycin in 489 European American individuals and validated findings in three independent cohorts totaling 439 European American individuals. In primary analyses, the chromosome 6q22.31 locus was associated with increased serum creatinine levels while on vancomycin therapy (most significant variant rs2789047, risk allele A, β = -0.06, p = 1.1 x 10^-7). SNPs in this region had consistent directions of effect in the validation cohorts, with a meta-p of 1.1 x 10^-7. Variation in this region on chromosome 6, which includes the genes TBC1D32/C6orf170 and GJA1 (encoding connexin43), may modulate risk of vancomycin-induced kidney injury.     

The 6-azauridine analogues possessing sedative and hypnotic effects.

Pharmacological effects of 6-azauridine (4) analogues were evaluated using hypnotic activity, pentobarbital (PB)-induced sleep prolongation and locomotor activity as indices. Compound 4, N3-benzyl- (10), N3-o-xylyl- (11), N3-m-xylyl- (12), N3-p-xylyl- (13), N3-alpha-phenylethyl-substituted 6-azauridine (14) exhibited hypnotic activity and PB-induced sleep prolongation, whereas N3-alkyl substituted analogues (methyl-, ethyl-, n-propyl-, n-butyl- and allyl-substitution) did not. Compound 4 and xylyl analogues (11-13) significantly decreased locomotor activity of mice by i.c.v. injection and produced motor incoordination. The results indicate that 4 and its benzyl related analogues, but not alkyl analogues have depressant effects on the central nervous system (CNS).