Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.     

ALS2/alsin deficiency in neurons leads to mild defects in macropinocytosis and axonal growth

Loss of function mutations in the ALS2 gene account for a number of juvenile/infantile recessive motor neuron diseases, indicating that its gene product, ALS2/alsin, plays a crucial role in maintenance and survival for a subset of neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5 and is implicated in endosome dynamics in cells. However, the role of ALS2 in neurons remains unclear. To elucidate the neuronal ALS2 functions, we investigate cellular phenotypes of ALS2-deficient primary cultured neurons derived from Als2-knockout (KO) mice. Here, we show that ALS2 deficiency results not only in the delay of axon outgrowth in hippocampal neurons, but also in a decreased level of the fluid phase horseradish peroxidase (HRP) uptake, which represents the activity for macropinocytic endocytosis, in cortical neurons. Thus, ALS2 may act as a modulator in neuronal differentiation and/or development through regulation of membrane dynamics.     

Association of the PIK3C2G gene polymorphisms with type 2 DM in a Japanese population

The associations of five SNPs (SNPs1-5: A-5468G, A-3333G, C-1794T, C437T and T9148C) of the class II phosphoinositide 3-kinase γ-subunit (PIK3C2G) gene with type 2 diabetes were examined using a population of the Takahata Study (n (M/W): 2930 (1328/1602); age: 63.3 ± 10.2 years), a Japanese community-based study. Quantitative association study of the SNPs with HbA1c levels showed significant association for SNPs 2 and 4 (p = 0.018 and 0.004, respectively). A case-control association study of SNP 4 with diabetes by multiple logistic regression analysis showed a significant association of the genotype TT of the SNP with an odds ratio of 2.21 (p = 0.001) independently of age, gender and BMI. In the NGT subjects, serum fasting insulin levels in the at-risk genotype group of SNP 4 were significantly lower than those in the others (TT, TC, and CC, 4.9 ± 2.6, 5.4 ± 3.0, and 5.6 ± 3.4 μU/ml, respectively; p = 0.029).     

Production of cell-enclosing hollow-core agarose microcapsules via jetting in water-immiscible liquid paraffin and formation of embryoid body-like spherical tissues from mouse ES cells enclosed within these microcapsules

We developed agarose microcapsules with a single hollow core templated by alginate microparticles using a jet-technique. We extruded an agarose aqueous solution containing suspended alginate microparticles into a coflowing stream of liquid paraffin and controlled the diameter of the agarose microparticles by changing the flow rate of the liquid paraffin. Subsequent degradation of the inner alginate microparticles using alginate lyase resulted in the hollow-core structure. We successfully obtained agarose microcapsules with 20-50 microm of agarose gel layer thickness and hollow cores ranging in diameter from ca. 50 to 450 microm. Using alginate microparticles of ca. 150 microm in diameter and enclosing feline kidney cells, we were able to create cell-enclosing agarose microcapsules with a hollow core of ca. 150 microm in diameter. The cells in these microcapsules grew much faster than those in alginate microparticles. In addition, we enclosed mouse embryonic stem cells in agarose microcapsules. The embryonic stem cells began to self-aggregate in the core just after encapsulation, and subsequently grew and formed embryoid body-like spherical tissues in the hollow core of the microcapsules. These results show that our novel microcapsule production technique and the resultant microcapsules have potential for tissue engineering, cell therapy and biopharmaceutical applications.     

Docosahexaenoic acid disrupts in vitro amyloid β1‐40 fibrillation and concomitantly inhibits amyloid levels in cerebral cortex of Alzheimer’s disease model rats

We have previously reported that dietary docosahexaenoic acid (DHA) improves and/or protects against impairment of cognition ability in amyloid beta_1‐40 (Aβ_1‐40)‐infused Alzheimer’s disease (AD)‐model rats. Here, after the administration of DHA to AD model rats for 12 weeks, the levels of Aβ_1‐40, cholesterol and the composition of fatty acids were investigated in the Triton X100‐insoluble membrane fractions of their cerebral cortex. The effects of DHA on the in vitro formation and kinetics of fibrillation of Aβ_1‐40 were also investigated by thioflavin T fluorescence spectroscopy, transmission electron microscopy and fluorescence microscopy. Dietary DHA significantly decreased the levels of Aβ_1‐40, cholesterol and saturated fatty acids in the detergent insoluble membrane fractions of AD rats. The formation of Aβ fibrils was also attenuated by their incubation with DHA, as demonstrated by the decreased intensity of thioflavin T‐derived fluorescence and by electron micrography. DHA treatment also decreased the intensity of thioflavin fluorescence in preformed‐fibril Aβ peptides, demonstrating the anti‐amyloidogenic effects of DHA. We then investigated the effects of DHA on the levels of oligomeric amyloid that is generated during its in vitro transformation from monomers to fibrils, by an anti‐oligomer‐specific antibody and non‐reducing Tris‐Glycine gradient (4–20%) gel electrophoresis. DHA concentration‐dependently reduced the levels of oligomeric amyloid species, suggesting that dietary DHA‐induced suppression of in vivo Aβ_1‐40 aggregation occurs through the inhibitory effect of DHA on oligomeric amyloid species.     

Inhibition of interleukin-12 production by Trypanosoma brucei in rat macrophages

The immune response of a host infected with Trypanosoma brucei is modulated by trypomastigotes. We examined the changes in cytokine production in T. brucei gambiense (Wellcome strain; WS) infected rats and the influence on production of interleukin (IL)-12 by macrophages. The blood concentration of interferon-gamma, tumor necrosis factor-alpha, and IL-10 increased beginning the second day after infection. However, an increase in IL-12p40 was not observed until 4 days after infection. When spleen macrophages and Kupffer cells harvested from uninfected rats and HS-P cells (a rat macrophagelike cell line) were cocultured with WS, IL-12p40 production did not change. When HS-P cells were cultured with WS, transport of nuclear factor-kappaB into the nucleus increased. Levels of macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor mRNA in the spleens and livers of WS-infected rats were high in comparison with uninfected rats, suggesting that the WS promotes macrophage proliferation. The level of IL-12p40 mRNA in HS-P cells cocultured with WS increased in response to transfection with a small interfering RNA against M-CSF or addition of anti-M-CSF antibody. These results suggest that the WS inhibits IL-12p40 mRNA production by promoting production of macrophage colony-stimulating factor by macrophages.     

Compatibility among polymerase subunit proteins is a restricting factor in reassortment between equine H7N7 and human H3N2 influenza viruses

Reassortment is an important driving force for influenza virus evolution, and a better understanding of the factors that affect this process could improve our ability to respond to future influenza pandemics and epidemics. To identify factors that restrict the generation of reassortant viruses, we cotransfected human embryonic kidney cells with plasmids for the synthesis of viral RNAs of both A/equine/Prague/1/56 (Prague; H7N7) and A/Yokohama/2017/03 (Yokohama; H3N2) viruses together with the supporting protein expression plasmids. Of the possible 256 genotypes, we identified 29 genotypes in 120 randomly plaque-picked reassortants examined. Analyses of these reassortants suggested that the formation of functional ribonucleoprotein (RNP) complexes was a restricting factor, a finding that correlated with the activities of RNP complexes composed of different combinations of the proteins from the two viruses, as measured in a minigenome assay. For at least one nonfunctional RNP complex (i.e., Prague PB2, Prague PB1, Yokohama PA, and Prague NP), the lack of activity was due to the inability of the three polymerase subunit proteins to form a heterotrimer. Adaptation of viruses possessing a gene encoding a chimera of the PA proteins of the two viruses and the remaining genes from Prague virus resulted in compensatory mutations in the PB2 and/or PA protein. These results indicate substantial incompatibility among the gene products of the two test viruses, a critical role for the RNP complex in the generation of reassortant viruses, and a functional interaction of PB2 and PA.     

Rescue of mutant alpha-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes

Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing alpha-galactosidase A (alpha-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant alpha-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type alpha-Gal A was observed when microsomal vesicles containing wild-type or R301Q alpha-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of alpha-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type alpha-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.     

NO and CO binding profiles of hemoglobin vesicles as artificial oxygen carriers

Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O(2) and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected.     

Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs

We examined whether Siglec-9 modulates cytokine production in the macrophage cell line RAW264. Cells expressing Siglec-9 produced low levels of tumor necrosis factor (TNF)-α upon stimulation with lipopolysaccharide, peptidoglycan, unmethylated CpG DNA, and double-stranded RNA. On the other hand, interleukin (IL)-10 production was strongly enhanced in Siglec-9-expressing cells. Similar activities were also exhibited by Siglec-5. However, the up-regulation of IL-10 as well as the down-regulation of TNF-α was abrogated when two tyrosine residues in the cytoplasmic tail of Siglec-9 were mutated to phenylalanine. A membrane proximal ITIM mutant of Siglec-9 did not enhance IL-10 production but partly inhibited TNF-α production, indicating diverse regulation mechanisms of TNF-α and IL-10. Siglec-9 also enhanced the production of IL-10 in the human macrophage cell line THP-1. These results demonstrate that Siglec-9 enhances the production of the anti-inflammatory cytokine IL-10 in macrophages.