Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.     

Tumor Necrosis Factor (TNF)-α-induced Repression of GKAP42 Protein Levels through cGMP-dependent Kinase (cGK)-Iα Causes Insulin Resistance in 3T3-L1 Adipocytes

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-α, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-Iα, which bound to GKAP42, in these changes. cGK-Iα knockdown partially rescued TNF-α-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-α-induced repression of GKAP42 via cGK-Iα caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-α-induced pathway, cGK-Iα-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.     

Deletion of the adenosine A2A receptor increases the survival rate in a mice model of polymicrobial sepsis

Purinergic Signalling - We aim to investigate the role of A2A receptor in peritonitis-related sepsis by injection of a fecal solution (FS) as a model of polymicrobial infection. C57/black J6...     

Molecular identification of Taenia spp. in wolves (Canis lupus), brown bears (Ursus arctos) and cervids from North Europe and Alaska

Taenia tapeworms of Finnish and Swedish wolves (Canis lupus) and Finnish brown bears (Ursus arctos), and muscle cysticerci of Svalbard reindeer (Rangifer tarandus platyrhynchus), Alaskan Grant's caribou (Rangifer tarandus granti) and Alaskan moose (Alces americanus) were identified on the basis of the nucleotide sequence of a 396 bp region of the mitochondrial cytochrome c oxidase subunit 1 gene. Two species were found from wolves: Taenia hydatigena and Taenia krabbei. The cysticerci of reindeer, caribou and one moose also represented T. krabbei. Most of the cysticercal specimens from Alaskan moose, however, belonged to an unknown T. krabbei-like species, which had been reported previously from Eurasian elks (Alces alces) from Finland. Strobilate stages from two bears belonged to this species as well. The present results suggest that this novel Taenia sp. has a Holarctic distribution and uses Alces spp. as intermediate and ursids as final hosts.     

Centimetre-scale vertical variability of phenoxy acid herbicide mineralization potential in aquifer sediment relates to the abundance of tfdA genes

Centimetre-scale vertical distribution of mineralization potential was determined for 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) by 96-well microplate radiorespirometric analysis in aquifer sediment sampled just below the groundwater table. Mineralization of 2,4-D and MCPA was fastest in sediment samples taken close to the groundwater table, whereas only minor mineralization of MCPP was seen. Considerable variability was exhibited at increasing aquifer depth, more so with 2,4-D than with MCPA. This suggests that the abundance of MCPA degraders was greater than that of 2,4-D degraders, possibly due to the fact that the overlying agricultural soil had long been treated with MCPA. Mineralization of 2,4-D and MCPA was followed by increased abundance of tfdA class I and class III catabolic genes, which are known to be involved in the metabolism of phenoxy acid herbicides. tfdA class III gene copy number was approximately 100-fold greater in samples able to mineralize MCPA than in samples able to mineralize 2,4-D, suggesting that tfdA class III gene plays a greater role in the metabolism of MCPA than of 2,4-D. Degradation rate was found to correlate positively with tfdA gene copy number, as well as with the total organic carbon content of the sediment.     

Functional recruitment of the human complement inhibitor C4BP to Yersinia pseudotuberculosis outer membrane protein Ail

Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the pathogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control protein domain, we observed that complement control protein domains 6-8 are important for binding to Ail. Binding of C4BP was unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement.     

Expression and characterization of biologically active human Fas ligand produced in CHO cells

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 μg/L × 10⁶ cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays. S. Zappitelli and L. D’Alatri contributed equally to this work.     

Impact of mobile clinical analyzers on disaster medicine: a lesson from crush syndrome in the 1995 Hanshin-Awaji earthquake

The Great Hanshin-Awaji earthquake caused many people to develop crush syndrome. Analysis of these patients revealed that the severity is not related to their hemodynamics but to hematocrit, base deficits, and potassium concentrations soon after their extrications. In the disaster site, these parameters can only be measured using whole-blood samples by mobile instruments. The present study was made to evaluate the possibility of uses of a mobile measuring device, ABL77, for the triage of crush syndrome patients in disaster sites. Heparin-added blood samples and serum samples were collected from patients admitted to Senshu Critical Care Medical Center. Blood gases, electrolytes, and hematocrit of the heparin-added blood samples were measured using ABL77 and compared with those measured using the stationary device ABL725. Potassium concentrations of the heparin-added blood samples measured by the ABL77 were compared with those of the serum samples measured by the stationary EA06T. Significant correlations were observed between the measurements. We conclude that the ABL77 was satisfactorily compatible with stationary devices in the hospital. Medical institutions should have simplified, mobile measuring devices as a precaution against disasters, so that they can get ready to take appropriate action promptly.