Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β

In a previous study, the Notch pathway inhibited with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (also called DAPT) was shown to promote the differentiation of fetal liver stem/progenitor cells (FLSPCs) into hepatocytes and to impair cholangiocyte differentiation. The precise mechanism for this, however, was not elucidated. Two mechanisms are possible: Notch inhibition might directly up-regulate hepatocyte differentiation via HGF (hepatocyte growth factor) and HNF (hepatocyte nuclear factor)-4α or might impair cholangiocyte differentiation thereby indirectly rendering hepatocyte differentiation as the dominant state. In this study, HGF and HNF expression was detected after the Notch pathway was inhibited. Although our initial investigation indicated that the inhibition of Notch induced hepatocyte differentiation with an efficiency similar to the induction via HGF, the results of this study demonstrate that Notch inhibition does not induce significant up-regulation of HGF or HNF-4α in FLSPCs. This suggests that Notch inhibition induces hepatocyte differentiation without the influence of HGF or HNF-4α. Moreover, significant down-regulation of HNF-1β was observed, presumably dependent on an impairment of cholangiocyte differentiation. To confirm this presumption, HNF-1β was blocked in FLSPCs and was followed by hepatocyte differentiation. The expression of markers of mature cholangiocyte was impaired and hepatocyte markers were elevated significantly. The data thus demonstrate that the inhibition of cholangiocyte differentiation spontaneously induces hepatocyte differentiation and further suggest that hepatocyte differentiation from FLSPCs occurs at the expense of the impairment of cholangiocyte differentiation, probably being enhanced partially via HNF-1β down-regulation or Notch inhibition.     

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris–HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65 °C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.     

Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C₄ plants.     

Aquaporins in development – a review

Water homeostasis during fetal development is of crucial physiologic importance. It depends upon maternal fetal fluid exchange at the placenta and fetal membranes, and some exchange between fetus and amniotic fluid can occur across the skin before full keratinization. Lungs only grow and develop normally with fluid secretion, and there is evidence that cerebral spinal fluid formation is important in normal brain development. The aquaporins are a growing family of molecular water channels, the ontogeny of which is starting to be explored. One question that is of particular importance is how well does the rodent (mouse, rat) fetus serve as a model for long-gestation mammals such as sheep and human? This is particularly important for organs such as the lung and the kidney, whose development before birth is very much less in rodents than in the long-gestation species.     

Enhanced oxidative stress impairs cAMP-mediated dilation by reducing Kv channel function in small coronary arteries of diabetic rats

We have shown that short-term exposure of rat small coronary arteries (RSCAs) to high glucose enhances superoxide (O2-*) formation and impairs cAMP-mediated dilation by reducing voltage-gated K+ (Kv) channel function. However, it is not clear whether the impairment also occurs in diabetes mellitus (DM), where alternate mechanisms could mask or aggravate vasodilator dysfunction. RSCAs were isolated from control and streptozotocin-induced diabetic rats. Reduced constriction to 4-aminopyridine (4-AP) was observed in RSCAs from DM rats, indicating Kv channel impairment. Forskolin increased 4-AP-inhibitable K+ channel open-state probability and whole cell K+ current density in coronary myocytes from non-DM rats but had little effect on K+ current density in cells from DM rats. Diminished dilation to 8-bromo-cAMP, forskolin, or isoproterenol was observed in DM RSCAs. The attenuated dilation to forskolin or isoproterenol in DM RSCAs was partially restored by application of the superoxide dismutase mimetic manganese[III] tetrakis (4-benzoic acid) porphyrin. Histofluorescence studies using hydroethidine revealed a blockage of O2-* generation by the NADPH oxidase inhibitor apocynin in DM RSCAs. Sepiapterin, a precursor of tetrahydrobiopterin, had little effect on hyperglycemia-induced O2-* formation. Consistent with the findings from the concurrent fluorescence study, apocynin also partially restored the reduced dilator response to forskolin in DM RSCAs. Forskolin-induced cAMP production was unaltered in DM. We conclude that in diabetes, enhanced O2-* formation by activation of NADPH oxidase impairs cAMP-medicated dilation in RSCAs by inhibiting Kv channel activity.     

A ‘Chinese Spring’ wheat (Triticum aestivum L.) bacterial artificial chromosome library and its use in the isolation of SSR markers for targeted genome regions

A bacterial artificial chromosome (BAC) library was constructed from the bread wheat (Triticum aestivum L.) genotype ‘Chinese Spring’ (‘CS’). The library consists of 395,136 clones with an estimated average insert size of 157 kb. This library provides an estimated 3.4-fold genome coverage for this hexaploid species. The genome coverage was confirmed by RFLP analysis of single-copy RFLP clones. The CS BAC library was used to develop simple sequence repeat (SSR) markers for targeted genome regions using five sequence-tagged-site (STS) markers designed from the chromosome arm of 3BS. The SSR markers for the targeted genome region were successfully obtained. However, similar numbers of new SSR markers were also generated for the other two homoeologous group 3 chromosomes. This data suggests that BAC clones belonging to all three chromosomes of homoeologous group 3 were isolated using the five STS primers. The potential impacts of these results on marker isolation in wheat and on library screening in general are discussed.     

Genetic characterization and molecular mapping of Hessian fly resistance genes derived from Aegilops tauschii in synthetic wheat

Two synthetic hexaploid wheat lines (×Aegilotriticum spp., 2n = 6x = 42, genomes AABBDD), SW8 and SW34, developed from the crosses of the durum wheat cultivar Langdon (Triticum turgidum L. var. durum, 2n = 4x = 28, genomes AABB) with two Aegilops tauschii Cosson accessions (2n = 2x = 14, genome DD), were determined to carry Hessian fly [Mayetiola destructor (Say)] resistance genes derived from the Ae. tauschii parents. SW8 was resistant to the Hessian fly biotype Great Plains (GP) and strain vH13 (virulent to H13). SW34 was resistant to biotype GP, but susceptible to strain vH13. Allelism tests indicated that resistance genes in SW8 and SW34 may be allelic to H26 and H13 or correspond to paralogs at both loci, respectively. H26 and H13 were localized to chromosome 4D and 6D, respectively, in previous studies. Molecular mapping in the present study, however, assigned the H26 locus to chromosome 3D rather than 4D. On the other hand, mapping of the resistance gene in SW34 verified the previous assignment of the H13 locus to chromosome 6D. Linkage analysis and physical mapping positioned the H26 locus to the chromosomal deletion bin 3DL3-0.81–1.00. A linkage map for each of these two resistance genes was constructed using simple sequence repeat (SSR) and target region amplification polymorphism (TRAP) markers.     

Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase/N-sulfotransferase 2

Heparin and heparan sulfate are linear sulfated polysaccharides that exert a multitude of biological functions. Heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase isoform 2 (NDST-2), a key enzyme in the biosynthesis of heparin, contains two distinct activities. This bifunctional enzyme removes the acetyl group from N-acetylated glucosamine (N-deacetylase activity) and transfers a sulfuryl group to the unsubstituted amino position (N-sulfotransferase activity). The N-sulfotransferase activity of NDST has been unambiguously localized to the C-terminal domain of NDST. Here, we report that the N-terminal domain of NDST-2 retains N-deacetylase activity. The N-terminal domain (A66-P604) of human NDST-2, designated as N-deacetylase (NDase), was cloned as a (His)(6)-fusion protein, and protein expression was carried out in Escherichia coli. Heparosan treated with NDase contains N-unsubstituted glucosamine and is highly susceptible to N-sulfation by N-sulfotransferase. Our results conclude that the N-terminal domain of NDST-2 contains functional N-deacetylase activity. This finding helps further elucidate the mechanism of action of heparan sulfate N-deacetylase/N-sulfotransferases and the biosynthesis of heparan sulfate in general.     

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.     

Overcoming original antigenic sin to generate new CD8 T cell IFN-gamma responses in an antigen-experienced host

The failure to mount effective immunity to virus variants in a previously virus-infected host is known as original antigenic sin. We have previously shown that prior immunity to a virus capsid protein inhibits induction by immunization of an IFN-gamma CD8+ T cell response to an epitope linked to the capsid protein. We now demonstrate that capsid protein-primed CD4+ T cells secrete IL-10 in response to capsid protein presented by dendritic cells, and deviate CD8+ T cells responding to a linked MHC class I-restricted epitope to reduce IFN-gamma production. Neutralizing IL-10 while delivering further linked epitope, either in vitro or in vivo, restores induction by immunization of an Ag-specific IFN-gamma response to the epitope. This finding demonstrates a strategy for overcoming inhibition of MHC class I epitopes upon immunization of a host already primed to Ag, which may facilitate immunotherapy for chronic viral infection or cancer.