Detection of SS-A/Ro and SS-B/La Autoantibodies Using Immunoblotting Procedure and Characterization of the Antigens from 293 Cells and KB Cells

Specific and sensitive assay was performed to detect both antiSS-A/Ro and antiSS-B/La antibodies in sera of patients with collagen diseases including SLE, PSS, etc. The SS-A/Ro and SS-B/La antigens were prepared from human spleen (HSE) and cultured human cell line (KB cells), while rabbit thymus extract (RTE) was used as SS-B/La antigen marker. The antigens were partially purified by DEAE cellulose column chromatography. The SS-A/Ro antibody was shown to react mainly with 58KDa peptide by means of immunoblotting. Sera containing both the SS-A/Ro and SS-B/La antibody reacted with 40KDa peptide of RTE, and 58KDa, 42KDa and 40KDa peptides of HSE. We found that some of SS-A/Ro antisera could further react with 64KDa peptide in HSE. The 58KDa peptide is rich in a cytoplasmic fraction of KB cells, and the 40KDa peptide in the nucleoplasmic fraction. KB cells are not less good source of the antigens than human spleen. Extracts of 293 cells (human embryonic kidney cells expressing adenovirus-5 El gene) were prepared by the same method from KB cells, though immunoblotting patterns of both SS-A/Ro and SS-B/La antigens of 293 cell extracts are similar to those of KB cells, the relative content of SS-B/La antigens in 293 cell extracts are decreased

Successful implantation after reducing matrix metalloproteinase activity in the uterine cavity

BACKGROUND: Recently, the concept of recurrent implantation failure (RIF) in assisted reproductive technology has been enlarged. Chronic uterine inflammation is a known cause of implantation failure and is associated with high matrix metalloproteinase (MMP) activity in uterine cavity flushing. MMP activity of women with RIF has been reported to be higher than that of fertile women. In the present retrospective study we evaluated the efficacy of treatment for high MMP activity in the uterine cavity of patients with RIF. METHODS: Of the 597 patients recruited to the study, 360 patients underwent MMP measurements and 237 patients did not (control group). All patients had failed to become pregnant, despite at least two transfers of good-quality embryos. Gelatinase MMP-2 and MMP-9 activity in uterine flushing fluid was detected by enzymology (MMP test). All samples were classified into two groups (positive or negative) based on the intensity of the bands on the enzyme zymogram, which represents the degree of MMP activity. Patients who tested positive on the initial test were treated for 2 weeks with a quinolone antibiotic and a corticosteroid, and subsequently underwent a second MMP test. Negative results on the second MMP tests after treatment and subsequent rates of pregnancy and miscarriage were used to evaluate the efficacy of treatment. Data were analyzed by the Mann–Whitney U-test and the chi-square test. RESULTS: Of the patients who underwent the MMP test, 15.6% had positive results (high MMP activity). After treatment, 89.3% of patients had negative results on the second MMP test. These patients had a significantly better pregnancy rate (42.0%) than the control group (26.6%), as well as a lower miscarriage rate (28.5% vs 36.5%, respectively). CONCLUSIONS: A 2-week course of antibiotics and corticosteroids effectively improves the uterine environment underlying RIF by reducing MMP activity

Structural Basis for Receptor-Mediated Selective Autophagy of Aminopeptidase I Aggregates

SummarySelective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity

Changes in the Collagen Binding Property of Human Plasma Fibronectin with Aging

We investigated changes in quality of plasma fibronectin with aging, especially in the interaction of fibronectin with collagen, using an enzyme-linked immunosorbent assay. When fibronectin isolated from the plasma of young people was compared with that from old people, the latter was found to bind poorly to gelatin, Type I and Type III collagens. No differences in molecular weight were detected between the two types of fibronectin by SDS-polyacrylamide gel electrophoresis

Effect of Hydrogen Peroxide and High Glucose on the Glucose Metabolism of Lymphoma-derived U937 Cells

Our study aimed to clarify specific oxidative stress and glucose metabolic disorders in hemodialysis patients, by examining hydrogen peroxide (H2O2) - and high glucose-induced oxidative stress, glucose transport and the failure of glycolysis. As an in vitro blood cell model of end-stage renal disease (ESRD) in patients with diabetes, human monocytic U937 cells of malignant lymphoma origin were exposed to high glucose (28.9mM) for 6 days, with 5mM H2O2 added on the last day. The generation of intracellular reactive oxygen species (ROS), glucose levels, lactate levels, AMP-activated protein kinase (AMPK) activity and Glut4 levels were examined. Exposure of U937 cells to H2O2 resulted in a significant increase in intracellular ROS generation and glucose levels. Under high glucose conditions, treatment with H2O2 significantly promoted these actions. In H2O2-induced U937 cells, AMPK activity and Glut4 levels were significantly increased, but lactate and pyruvate levels were significantly decreased. Thus, exposure of U937 cells to H2O2 and a high glucose load promoted an increase in intracellular ROS, and exposure to H2O2 induced increased glucose transport and high intracellular glucose due to reduced glycolytic metabolism. This suggests that reduced glycolytic metabolism might be induced in states of high oxidative stress in hemodialysis patients with diabetes

Common Variants in MAGI2 Gene Are Associated with Increased Risk for Cognitive Impairment in Schizophrenic Patients

Schizophrenia is a complex psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive impairment. MAGI2, a relatively large gene (∼1.5 Mbps) that maps to chromosome 7q21, is involved in recruitment of neurotransmitter receptors such as AMPA- and NMDA-type glutamate receptors. A genetic association study designed to evaluate the association between MAGI2 and cognitive performance or schizophrenia has not been conducted. In this case-control study, we examined the relationship of single nucleotide polymorphism (SNP) variations in MAGI2 and risk for schizophrenia in a large Japanese sample and explored the potential relationships between variations in MAGI2 and aspects of human cognitive function related to glutamate activity. Based on the result of first schizophrenia genome-wide association study in a Japanese population (JGWAS), we selected four independent SNPs and performed an association study using a large independent Japanese sample set (cases 1624, controls 1621). Wisconsin Card Sorting Test (WCST) was used to evaluate executive function in 114 cases and 91 controls. We found suggestive evidence for genetic association of common SNPs within MAGI2 locus and schizophrenia in Japanese population. Furthermore in terms of association between MAGI2 and cognitive performance, we observed that genotype effect of rs2190665 on WCST score was significant (p = 0.034) and rs4729938 trended toward significance (p = 0.08). In conclusion, although we could not detect strong genetic evidence for association of common variants in MAGI2 and increased schizophrenia risk in a Japanese population, these SNPs may increase risk of cognitive impairment in schizophrenic patients

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study