O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1^{R245A knock-in}), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1^{R245A knock-in} and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein''s functions.     

Preparation and immunological characterization of β-lactoglobulin-amylose conjugate

β-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.     

Effects of struvite formation and nitratation promotion on nitrogenous emissions such as NH3, N2O and NO during swine manure composting

To reduce nitrogenous emissions from composting, two different countermeasures were applied simultaneously in swine manure composting. One was forming struvite by adding Mg and P at the start of composting, and the other was to promote nitratation (nitrite being oxidized nitrate) by adding nitrite-oxidizing bacteria after the thermophilic phase of composting. In the laboratory- and mid-scale composting experiments, 25-43% of NH3, 52-80% of N2O and 96-99% of NO emissions were reduced. From the nitrogen balance, it was revealed that the struvite formation reduced not only NH3, but also other nitrogenous emissions except N2O. The amount of total nitrogen losses was reduced by 60% by the two combined countermeasures, against 51% by the struvite formation alone. However, the nitratation promotion dissolved struvite crystals due to the pH decline, diminishing the effect of struvite as a slow-release fertilizer.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Different Impacts of Neck Circumference and Visceral Obesity on the Severity of Obstructive Sleep Apnea Syndrome

Our aim was to investigate the significance of neck circumference (NC) on the presence and severity of obstructive sleep apnea (OSA) syndrome independent of visceral fat (VF) obesity. A total of 219 subjects with suspected OSA underwent a complete polysomnography (PSG) study, along with the measurement of NC, and total body fat (TF) and VF levels (VFLs) measured by bioelectrical impedance analysis. We proposed NC divided by height (NC/H) as the simple index for height‐corrected NC in Japanese subjects. NC/H exhibited a significantly stronger correlation than NC per se with BMI (r = 0.781 vs. 0.675, P = 0.0178), TF (r = 0.531 vs. 0.156, P < 0.0001), and VF (r = 0.819 vs. 0.731, P = 0.0203), indicating that NC/H is a better indicator of visceral obesity than NC per se. Interestingly, despite the strong correlation between NC/H and VFL, VFL was significantly associated with the apnea‐hypopnea index (AHI) ≥5, ≥15, and ≥30, but not with ≥40 or ≥50, whereas NC/H was significantly associated with higher AHI values, i.e., AHI ≥50 but not with lower AHI value. Furthermore, multiple regression analyses revealed that VFL and NC/H were independently associated with the square root of AHI (AHI^0.5) levels in obese and nonobese patients, respectively. In conclusion, NC is associated with the severity of OSA independently of visceral obesity, especially in nonobese patients.     

Selenoureas and thioureas are effective superoxide radical scavengers in vitro

Oxygen radicals, such as superoxide radicals, embellishing DNA, protein, lipids, etc., and carrying out the obstacle of the function of a cell is known. It depends for the oxidant level in the living body on the balance of a generation system and an elimination system of oxygen radicals, and research which controls an oxidant level in the living body is briskly done by taking in the substance which eliminates an oxygen radical. We investigated scavenging effects of superoxide radicals by selenoureas and thioureas using a highly sensitive and quantitative chemiluminescence method. At 330 nM, five selenoureas and five thioureas scavenged fractions of superoxide radicals (O2-) ranging from 8.4% to 87.6%. Among five N,N-unsubstituted selenoureas and N,N-unsubstituted thioureas 1-selenocarbamoylpiperidine and 1-thiocarbamoylpyrrolidine were the most effective scavengers. A possibility that selenoureas could use it as a new superoxide anion-scavenging substance from the result of this research became clear.     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.