Late preconditioning by ethanol is initiated via an oxidant-dependent signaling pathway

Ingestion of alcoholic beverages at low to moderate levels 24 h prior to ischemia and reperfusion (I/R) prevents postischemic leukocyte/endothelial cell adhesive interactions, a phenomenon referred to as late ethanol preconditioning (EtOH-PC). The aim of this study was to determine whether oxidants act as initiators of late EtOH-PC. Ethanol was instilled into the stomachs of C57BL/6 mice as a bolus by gavage at a dose that produced a peak plasma concentration of 45 mg/dl 30 min after administration and returned to control levels 60 min after ingestion. Twenty four hours later, the superior mesenteric artery was occluded for 45 min followed by 70 min of reperfusion. The numbers of rolling and firmly adherent leukocytes were quantified in postcapillary venules of the small intestine in sham animals (no EtOH-PC, no I/R), in mice subjected to I/R alone or EtOH-PC + I/R, and in animals treated with Mn-TBAP (a cell-permeant superoxide dismutase mimetic), oxypurinol (a XO inhibitor), the NAD(P)H oxidase inhibitors PR-39 or apocynin, or oxypurinol plus PR39 during the period of EtOH-PC on Day 1 followed by I/R on Day 2. In separate groups of mice, oxypurinol or apocynin were also administered 1 h after ethanol ingestion on Day 1, with induction of I/R 24 h later. I/R induced marked increases in leukocyte rolling and adherence, effects that were completely prevented by EtOH-PC. Coincident treatment with Mn-TBAP, oxypurinol, PR-39, apocynin, or oxypurinol plus PR-39 with ethanol attenuated these anti-inflammatory actions of EtOH-PC. However, administration of oxypurinol or apocynin 1 h after ethanol ingestion failed to prevent these protective effects of EtOH-PC. Our results indicate that reactive oxygen species formed during the period of ethanol exposure on Day 1 trigger the development of an anti-inflammatory phenotype that renders the small bowel resistant to the proadhesive effects of I/R 24 h later.     

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.     

Characteristics of Attempted Suicide by Patients with Schizophrenia Compared with Those with Mood Disorders: A Case-Controlled Study in Northern Japan

Recent reports suggest a lifetime suicide risk for schizophrenia patients of approximately 5%. This figure is significantly higher than the general population suicide risk consequently, detection of those at risk is clinically important. This study was undertaken to define the characteristics of suicide attempts by schizophrenia patients compared with attempts by patients with mood disorders. All patients were diagnosed using the ICD-10 criteria. The study population comprised 65 patients with F2 disorders (schizophrenia, schizotypal and delusional disorders), i.e., “the F2 group”, and 94 patients with F3 disorders (mood disorders), i.e., “the F3 group”, who presented in the clinical setting of consultation-liaison psychiatry. The F2 group had a significantly younger mean age and significantly higher ratios of ‘past/present psychiatric treatment’ and ‘more than 3 months interruption of psychiatric treatment’. In contrast, the ratios of ‘physical disorder comorbidity’, ‘alcohol intake at suicide attempt’ and ‘suicide note left behind’ were significantly higher in the F3 group. The F2 group attempted suicide by significantly more serious methods. Furthermore, ‘hallucination-delusion’ was the most prevalent motive in the F2 group and was the only factor that showed a significant association with the seriousness of the method of suicide attempt (OR = 3.36, 95% CI: 1.05–11.33).     

Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.     

Knock Out of S1P3 Receptor Signaling Attenuates Inflammation and Fibrosis in Bleomycin-Induced Lung Injury Mice Model

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.     

ESCRT-0 Protein Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (Hrs) Is Targeted to Endosomes Independently of Signal-transducing Adaptor Molecule (STAM) and the Complex Formation with STAM Promotes Its Endosomal Dissociation

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.     

Construction of a high-throughput rat genetic mapping system with 466 arbitrarily primed-representational difference analysis markers

Linkage mapping of quantitative trait loci (QTLs) requires genetic markers that can be efficiently genotyped for a large number of individuals. To isolate genetic markers suitable for this purpose, we previously established the arbitrarily primed RDA (AP-RDA) method. Dot-blotting AP-PCR products (AP-amplicons) onto filters at a high density and hybridization of the filters with the AP-RDA markers made it possible to genotype a large number of individuals simultaneously for multiple loci. In this study, by using 25 primers or primer combinations, we isolated a total of 419 AP-RDA markers by subtracting the AP-amplicon of BUF rats from that of ACI rats, and vice versa. By combining 47 previously isolated markers, a rat genetic map was drawn with 466 AP-RDA markers. Between two given strains of rats other than ACI and BUF, the average informativeness of the markers was 38%. As for the intercross of ACI and BUF rats, 12 selected primers served to genotype 259 loci. In addition, the amounts and quality of genomic DNA to be used for AP-PCR were examined to guarantee reliable genotyping. Now, initial genome scanning of the rat for linkage analysis can be performed efficiently using this mapping system with AP-RDA markers. Received: 28 April 2000 / Accepted: 14 June 2000     

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.     

Bronchioalveolar morphogenesis of human bronchial epithelial cells depending upon hepatocyte growth factor

Lung alveolar regeneration occurs in adult human lungs as a result of proliferation, differentiation and alveolar morphogenesis of stem cells. It is increasingly being believed that bronchial epithelial cells (BECs) have a potential as stem cells, because they are potent to differentiate into multiple central and peripheral lung cell types in three‐dimensional (3D) cultures, and they develop multiple foci with well‐differentiated histogenesis after transformed into neoplastic cells. In this study, we investigated morphogenic abilities of HBE135 human BECs immortalized by E6/E7 oncogene in 3D cultures. When HBE135 cells were cultured alone or co‐cultured with endothelial cells, the cells formed spherical colonies without branching. However, in co‐culture with lung fibroblast MRC‐9 cells, HBE135 cells formed colonies with bronchioalveolar‐like complex branching, suggesting that MRC‐9‐derived soluble factor(s) are responsible for the branching formation. MRC‐9 cells, not endothelial cells, were found to highly express hepatocyte growth factor (HGF), a soluble molecule involved in liver and kidney regeneration. An anti‐HGF neutralizing antibody severely suppressed the complex branching formation, but addition of HGF could not sufficiently compensate the morphogenic effects of MRC‐9 cells, suggesting that MCR‐9‐derived HGF was necessary but insufficient for the bronchioalveolar structure formation. Immunohistochemistry revealed that Met, a cognate receptor for HGF, was highly expressed and phosphorylated in neoplastic BECs from lung adenocarcinomas with well‐differentiated, not poorly differentiated, histogenesis. These results are consistent with the notion that BECs have an aspect of stem cells. This aspect appears to become manifest through HGF–Met signalling pathway activation.