Maternal Tn Immunization Attenuates Hyperoxia-Induced Lung Injury in Neonatal Rats Through Suppression of Oxidative Stress and Inflammation

Hyperoxia therapy is often required to treat newborns with respiratory disorders. Prolonged hyperoxia exposure increases oxidative stress and arrests alveolar development in newborn rats. Tn antigen is N-acetylgalactosamine residue that is one of the most remarkable tumor-associated carbohydrate antigens. Tn immunization increases the serum anti-Tn antibody titers and attenuates hyperoxia-induced lung injury in adult mice. We hypothesized that maternal Tn immunizations would attenuate hyperoxia-induced lung injury through the suppression of oxidative stress in neonatal rats. Female Sprague–Dawley rats (6 weeks old) were intraperitoneally immunized five times with Tn (50 μg/dose) or carrier protein at biweekly intervals on 8, 6, 4, 2, and 0 weeks before the day of delivery. The pups were reared in room air (RA) or 2 weeks of 85% O2, creating the four study groups: carrier protein + RA, Tn vaccine + RA, carrier protein + O2, and Tn vaccine + O2. The lungs were excised for oxidative stress, cytokine, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, and histological analysis on postnatal day 14. Blood was withdrawn from dams and rat pups to check anti-Tn antibody using western blot. We observed that neonatal hyperoxia exposure reduced the body weight, increased 8-hydroxy-2-deoxyguanosine (8-OHdG) expression and lung cytokine (interleukin-4), increased mean linear intercept (MLI) values, and decreased vascular density and VEGF and PDGF-B expressions. By contrast, Tn immunization increased maternal and neonatal serum anti-Tn antibody titers on postnatal day 14, reduced MLI, and increased vascular density and VEGF and PDGF-B expressions to normoxic levels. Furthermore, the alleviation of lung injury was accompanied by a reduction in lung cytokine and 8-OHdG expression. Therefore, we propose that maternal Tn immunization attenuates hyperoxia-induced lung injury in neonatal rats through the suppression of oxidative stress and inflammation

Phosphorylation of Ubc9 by Cdk1 Enhances SUMOylation Activity

Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9

Characterization of the Receptors for Luteinizing Hormone/Human Chorionic Gonadotropin and Lipoproteins in the Luteinized Rat Ovary.

The studies presented in this dissertation are concerned with the subunit structure of the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, the assembly of the hCG-receptor complex and the proterties of the lipoprotein receptors. To elucidate the subunit structure of the LH/hCG receptor, a cross-linker noncleavable by reducing agents, disuccinimidyl suberate, and a cleavable cross-linker, dithiobis(succinimidyl propionate), were employed to cross-link ('125)I-hCG with the LH/hCG receptor. The molecular weight of ('125)I-hCG-linked receptor complex and the receptor subunit structure were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by radioautography. Using this approach, the LH/hCG receptor was shown to be a disulfide-linked complex consisting of four nonidentical subunits with an apparent Mr of 230,000-260,000. To examine the relative spatial relationship of the hCG subunits in the assembly of the hCG-receptor complex, the efficacy of the (alpha) and (beta) subunits of hCG cross-linked to the LH/hCG receptor was tested. The autoradiographic profile of ('125)I-hCG-linked receptor complex with the label in the (alpha) subunit of hCG showed that the (alpha) subunit can cross-link with all four subunits of the LH/hCG receptor. The (beta) subunit of hCG, unlike the (alpha) subunit, can cross-link only weakly with the smallest subunit of the LH/hCG receptor. These results suggest that both (alpha) and (beta) subunits are intimately associated with the receptor, but the bulk of the (beta) subunit is buried by the (alpha) subunit. For the characterization of the properties of the lipoprotein receptors, the specific binding of ('125)I-labeled low density lipoprotein (LDL), high density lipoprotein (HDL), apolipoprotein AI(.)dimyristoyl phosphatidyl choline complex (ApoAI(.)DMPC), and ApoAII(.)DMPC to luteal cell membranes was examined. These membranes were found to have distinct receptors for LDL and HDL, with respect to their binding specificity, sensitivity to protease, calcium ion and ionic strength. Both LDL and HDL receptors can be induced in the ovary by in vivo hCG treatment. The HDL receptor was also demonstrated to specifically recognize ApoAI and ApoAII.Ph.D.BiophysicsUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/159953/1/8412164.pd

Apolipopcotein specificity of high density lipoprotein receptors in luteinized rat ovary

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24804/1/0000230.pd

Phosphorylation-dependent SUMOylation of the transcription factor NF-E2.

Nuclear factor erythroid-derived 2 (NF-E2), a heterodimer composed of p45 and p18, is a transcriptional activator in hematopoietic progenitors. The transcriptional activity of NF-E2 is not only upregulated by SUMOylation but also stimulated by the cAMP-dependent protein kinase A (PKA). However, the relationship between SUMOylation and phosphorylation in the activation of NF-E2 is unclear. In the present studies, we have demonstrated that PKA enhances NF-E2 SUMOylation in an in vitro system using purified proteins, suggesting a possible mechanism for PKA-dependent activation of the NF-E2 transcription factor through SUMOylation

Expression and purification of p45/NF-E2 heterodimer and p45.

<p>A) Whole cell lysates of baculovirus co-expressing p45 and p18 were subjected to Mono Q Sepharose anion-exchange chromatography. Proteins were eluted by a NaCl gradient in 50 mM Tris-HCl, pH 8.0 buffer. All isolated fractions (#1–24) were collected by FPLC. The fractions (#11–14) primarily contain the p45/p18 heterodimer. Fractions (#10–17) were subjected to Western blotting with antibody against p45 or p18. B) Eluates containing the p45/p18 heterodimer (fractions #11–14) were subjected to nickel affinity purification and proteins were eluted with 100 mM imidazole. Eluates containing the p45 monomer were similarly purified by nickel affinity column. The purified p45/p18 heterodimer and p45 monomer were resolved by 11.25% and 12.5% SDS-PAGE, respectively, and subsequently immunoblotted with p45 or p18 antibody as indicated.</p