In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.     

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.     

Comparison of a Stool Antigen Test and Serology for the Diagnosis of Helicobacter pylori Infection in Mass Survey

Background: Serum antibody to Helicobacter pylori is tested in mass screening for gastric cancer along with the level of serum pepsinogens (PG) I and II. Recently, stool antigen tests have been developed as a new non-invasive test. We examined H. pylori infection by both serology and stool antigen test in a mass survey and compared the results to estimate applicability of stool antigen test for mass survey.
Methods: A total of 994 healthy adults who received mass survey in April 2005 were tested. There were 379 men and 615 women, and the mean age was 57.7 years old. Stool samples were used to measure a H. pylori- specific antigen by enzyme immunoassay. Serum samples were tested for the prevalence of IgG antibody to H. pylori , and the level of PGs I and II was also measured to determine the presence of atrophic gastritis.
Results: Infection of H. pylori was defined as positive 61.4% and 56.4% by serology and stool antigen test, respectively. The concordance of both tests was not affected by gender and age of the subjects but difference was seen in subjects with atrophic gastritis. In particular, positivity of stool antigen test (81.8%) was significantly lower than that of serology (88.7%, p  < .05) in 303 subjects with severe atrophic gastritis.
Conclusions: Stool antigen test, which detects present but not previous infection of H. pylori , would be applicable to diagnose H. pylori infection in mass survey. Usefulness of stool antigen tests for the screening of gastric cancer should be examined.     

The Relationship Between Serum Selenium Concentration and Neutrophil Function in Peripheral Blood

We evaluated the relationships between neutrophil-related functions and serum selenium (Se) concentration in the general population. We examined 800 subjects who had participated in the Iwaki Health Promotion Project in 2005 to determine the relationships between serum Se concentration and neutrophil-related functions such as the production capability of reactive oxygen species (ROS), phagocytic activity, and serum opsonic activity (SOA). In nonstimulated neutrophils, i.e., in neutrophils at their baseline condition before the application of the phagocytic stimulus, the serum Se concentration tends to be high and the ROS production tends to be low. With regard to SOA, there was a significant negative correlation between lucigenin-dependent chemiluminescence and serum Se concentration in both men and women. Moreover, in women, a significant negative correlation was observed between luminol-dependent chemiluminescence and serum Se concentration. These results suggest that subjects with a lower serum Se concentration may be exposed to a greater chronic oxidative stress due to neutrophil ROS production. In addition, the findings of our study suggest that women rather than men benefit more from Se against oxidative stress.     

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.     

Development of a robust method for establishing B cell lines using Epstein-Barr Virus

B lymphoblastoid cell lines (B-LCLs) are generally established from B lymphocytes by infection with Epstein-Barr virus (EBV). As their genomic structure is stable in culture, B-LCLs are a valuable resource for many types of analysis. The efficiency of establishing B-LCLs from freshly obtained blood samples from healthy individuals is almost 100?%; however, for blood samples stored inappropriately after collection or held in long-term storage as peripheral blood mononuclear cells (PBMCs) in liquid nitrogen, the efficiency of B-LCL establishment can be considerably lower. To date, we have established more than 550 B-LCLs from 685 PBMC samples that have been stored in liquid nitrogen for over 20?yr. The PBMCs were prepared from blood samples donated by individuals belonging to native minority ethnic groups in outlying regions of South America and elsewhere. The establishment of B-LCLs from this material is difficult, and failure results in the waste of valuable and rare samples. We sought to improve our success rate for establishing B-LCLs from these difficult and irreplaceable samples by a detailed examination of each step of the process. The analysis showed that two parameters were particularly critical to the success rate: the density of the PBMCs plated after EBV infection and the EBV titer. These observations shed light on cases where establishment of B-LCLs was hard due to the small number of PBMCs or damage to the cells.     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.