EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1.

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EGF-domain-specific O-linked N-acetylglucosamine transferase (Eogt) permits mice conditionally lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at ~ 28 days after birth with greater loss of body weight, a greater increase in the number of goblet and Paneth cells, and greater downregulation of the Notch target gene Hes1, compared to Pofut1 deletion alone. These data reveal that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2.

Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[-/-] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[-/-]Slc35c2[-/-] mutants were indistinguishable in skeletal phenotype from Slc35c1[-/-] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[-/-]Slc35c2[-/-] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.

Glycans that regulate Notch signaling in the intestine.

Intestinal homeostasis is key to the maintenance of good health. The small intestine plays important roles in absorption, digestion, hormonal and immune functions. Crypt base columnar (CBC) stem cells residing at the bottom of crypts are nurtured by Paneth cells, and together create the stem cell niche, the foundation of intestinal homeostasis. CBC stem cells replicate to replenish their number, or differentiate into a variety of epithelial cells with specialized functions. Notch signaling is a cell-cell signaling pathway that regulates both the proliferation and differentiation of CBC stem cells. NOTCH1 and NOTCH2 stimulated by canonical Notch ligands DLL1 and DLL4 mediate Notch signaling in the intestine that, in concert with other signaling pathways including the WNT and BMP pathways, determines cell fates. Importantly, interactions between Notch receptors and canonical Notch ligands are regulated by O-glycans linked to Ser/Thr in epidermal growth factor-like (EGF) repeats of the Notch receptor extracellular domain (NECD). The O-glycans attached to NECD are key regulators of the strength of Notch signaling. Imbalances in Notch signaling result in altered cell fate decisions and may lead to cancer in the intestine. In this review, we summarize the impacts of mutations in Notch pathway members on intestinal development and homeostasis, with a focus on the glycosyltransferases that transfer O-glycans to EGF repeats of NOTCH1, NOTCH2, DLL1 and DLL4.

A novel hepatoprotective activity of Alangium salviifolium in mouse model.

In this study, the hepatoprotective activity of methanol bark extract of Alangium salviifolium (BEA) was evaluated for biochemical and histological parameters in swiss albino mice with CCl4-induced hepatotoxicity. The hepatomodulatory effect of two doses of BEA (20 and 50 mg/kg bw for 15 days by oral gavage) was assessed on antioxidant enzymes, phase I and phase II drug detoxifying enzymes. For the characterization of the extract, GC-MS analysis was performed that revealed the abundance of alkaloids and steroidal compounds. Total phenolic and flavonoid contents in BEA were 69.61 ± 0.18 mg GAE/g and 46.27 ± 3.44 mg Rutin/g, respectively. BEA administration decreased the levels of AST, ALT, and ALP, which were elevated due to hepatic damage by CCl4. BEA significantly decreased the lipid peroxidation, activities of LDH, and phase I enzymes including cytochrome P450 reductase, cytochrome b5 reductase while increased the activities of SOD, CAT, and phase II enzymes DT-diaphorase and glutathione S-transferase in liver. Further, histological evaluation of the liver tissue was suggestive of the protective effect of BEA against CCl4 toxicity. Together, these results suggest that BEA has strong hepatoprotective activity in mice which may also be attributed to its potential chemopreventive efficacy.

A highly precise cross-capacitive sensor for metal debris detection in insulating oil.

The presence of metal particles in lubricating oil produced during the wear and tear of mechanical equipment can harm its performance severely if not detected in time. Hence, the detection of such particles is necessary to predict and to prevent disastrous failures of the machines. This paper presents a new non-contact cross-capacitive sensor for the detection of metal particles in the lubricating oil. The sensor can detect each and every metal debris particle in the lubricating oil by monitoring the capacitance peak. The proposed capacitive sensor works on the principle of the Thompson-Lampard theorem. The sensor consists of four cylindrical electrodes with infinitesimal gaps wrapped around a hollow Teflon tube. The sensor has been modeled with finite element simulation software and then fabricated to verify the theory experimentally. Experimental results show that the capacitance value shows a sharp change in its value due to the presence of metal debris in the oil. The output of the sensor is highly precise (±0.82%) and accurate.

A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice.

BACKGROUND: Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. RESULTS: We now report that after backcrossing to C57BL/6 J mice for 11-15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice. CONCLUSION: We conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

Polyphenols of Salix aegyptiaca modulate the activities of drug metabolizing and antioxidant enzymes, and level of lipid peroxidation.

BACKGROUND: Salix aegyptiaca is known for its medicinal properties mainly due to the presence of salicylate compounds. However, it also contains other beneficial phytochemicals such as gallic acid, quercetin, rutin and vanillin. The aim of the study was to examine the redox potential, antioxidant and anti-inflammatory activity of these phytochemicals along with acetylsalicylic acid. METHODS: The redox potential and antioxidant activity of gallic acid, quercetin, rutin, vanillin and acetylsalicylic acid were determined by oxidation-reduction potential electrode method and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, respectively. In ex vivo studies, antioxidant activity of these phytochemicals was determined by lipid peroxidation and carbonyl content assay in the liver of mice. Anti-inflammatory activity was determined by protein denaturation method. Six-week old C57BL/6 mice treated with gallic acid (100 mg/kg body weight) and acetylsalicylic acid (25 and 50 mg/kg body weight) to investigate their in vivo modulatory effects on the specific activities of drug metabolizing phase I and phase II enzymes, antioxidant enzymes and level of lipid peroxidation in liver. RESULTS: The order of ability to donate electron and antioxidant activity was found to be: gallic acid > quercetin > rutin > vanillin > acetylsalicylic acid. In ex vivo studies, the similar pattern and magnitude of inhibitory effects of these phytochemicals against peroxidative damage in microsomes and protein carbonyl in cytosolic fraction were observed. In in vivo studies, gallic acid and acetylsalicylic acid alone or in combination, enhanced the specific activities of drug metabolizing phase I and phase II enzymes as well as antioxidant enzymes and also inhibited lipid peroxidation in liver. CONCLUSIONS: These findings show a close link between the electron donation and antioxidation potential of these phytochemicals, and in turn their biological activity. Gallic acid, quercetin, rutin and vanillin were found to be better electron donors and antioxidants and therefore, might be mainly responsible for the antioxidant properties of S. aegyptiaca, while acetylsalicylic acid provided its maximum anti-inflammatory activity.

Responsive Proteins in Wheat Cultivars with Contrasting Nitrogen Efficiencies under the Combined Stress of High Temperature and Low Nitrogen.

Productivity of wheat (Triticumaestivum) is markedly affected by high temperature and nitrogen deficiency. Identifying the functional proteins produced in response to these multiple stresses acting in a coordinated manner can help in developing tolerance in the crop. In this study, two wheat cultivars with contrasting nitrogen efficiencies (N-efficient VL616 and N-inefficient UP2382) were grown in control conditions, and under a combined stress of high temperature (32 °C) and low nitrogen (4 mM), and their leaf proteins were analysed in order to identify the responsive proteins. Two-dimensional electrophoresis unravelled sixty-one proteins, which varied in their expression in wheat, and were homologous to known functional proteins involved in biosynthesis, carbohydrate metabolism, energy metabolism, photosynthesis, protein folding, transcription, signalling, oxidative stress, water stress, lipid metabolism, heat stress tolerance, nitrogen metabolism, and protein synthesis. When exposed to high temperature in combination with low nitrogen, wheat plants altered their protein expression as an adaptive means to maintain growth. This response varied with cultivars. Nitrogen-efficient cultivars showed a higher potential of redox homeostasis, protein stability, osmoprotection, and regulation of nitrogen levels. The identified stress-responsive proteins can pave the way for enhancing the multiple-stress tolerance in wheat and developing a better understanding of its mechanism.