A potential model for studying the plasticity and reprogramming of human epidermal stem cells through preimplantation blastocyst microinjection

Microinjection of adult stem cells (ASCs) into blastocysts provides a classic model for studying ASC plasticity. To explore the molecular mechanisms that govern the reprogramming of ASCs, we evaluated the experimental model through microinjection of human epidermal stem cells (hEpiSCs) into mouse blastocysts. Mouse blastocysts underwent regular embryogenesis after microinjection of allogeneic cells, confirmed by morphological observation and embryo cell counting. hEpiSCs survive and integrate into mouse embryos, by monitoring the migration of injected cells at 2, 4, 12, 16 and 24 h. In this xenogeneic system, hEpiSCs could be reprogrammed within 24 h, as evidenced by the silencing of CK15 and Integrin beta 1 gene expression, without activation of Oct4 and Nanog. Microinjection of hEpiSCs into mouse blastocysts provides an efficient model for studying the molecular mechanisms of their plasticity. Moreover, the possibility of inducing pluripotent stem cells without transgenes or viruses can be entertained.     

Effects of cutinase on the enzymatic shrink-resist finishing of wool fabrics

A novel microbial cutinase from Thermobifida fusca WSH04 was applied in the pretreatment of wool fabrics followed by protease treatment, aiming at improving the wettability of the samples by hydrolyzing the outmost bound lipids in the wool surface. Cutinase pretreatment could increase the efficacy of the subsequent protease treatment by improving the wettability, dyeability, and shrink-resistance of the wool fabrics. The data obtained by the XPS method showed the changes of elemental concentration in the wool surface after cutinase pretreatment. Compared with the fabrics treated with hydrogen peroxide and protease, the combination of cutinase and protease treatments produced better results in terms of wettability and shrink-resistance with less strength loss. The anti-felting property of the fabrics treated with the enzymatic resist-shrink technique is very promising to meet the commercial standard.     

Plasmodium falciparum signal peptide peptidase is a promising drug target against blood stage malaria

The resistance of malaria parasites to current anti-malarial drugs is an issue of major concern globally. Recently we identified a Plasmodium falciparum cell membrane aspartyl protease, which binds to erythrocyte band 3, and is involved in merozoite invasion. Here we report the complete primary structure of P. falciparum signal peptide peptidase (PfSPP), and demonstrate that it is essential for parasite invasion and growth in human erythrocytes. Gene silencing suggests that PfSPP may be essential for parasite survival in human erythrocytes. Remarkably, mammalian signal peptide peptidase inhibitors (Z-LL)₂-ketone and L-685,458 effectively inhibited malaria parasite invasion as well as growth in human erythrocytes. In contrast, DAPT, an inhibitor of a related γ-secretase/presenilin-1, was ineffective. Thus, SPP inhibitors specific for PfSPP may function as potent anti-malarial drugs against the blood stage malaria.     

Antibacterial functionalization of wool fabric via immobilizing lysozymes

Greater attention has been given to enzymatic processes of textiles as effective alternatives to conventional chemical treatments because of the non-toxic and eco-friendly characteristics of enzymes as well as the increasingly important requirement for reducing pollution in textile production. A new functionalization method for wool fabrics based on immobilization of lysozymes was investigated in this paper. Wool fabric was first activated with glutaraldehyde, and then employed to covalently immobilize lysozymes. Main immobilization parameters were optimized in terms of the activity of immobilized enzyme. A high activity of the immobilized enzyme was obtained when the fabric was activated at 25 °C for 6 h in a bath containing with 0.2% of glutaraldehyde followed by the immobilization at 4 °C and pH 7.0 for 6 h with 5 g l⁻¹ lysozyme. The scanning electron microscopy and staining tests based on modified Coomassie protein assay (Bradford method) revealed that the lysozyme was fixed covalently on the wool fabric. Wool fabrics immobilizing lysozymes presented a higher ratio of bacteriostasis to Staphylococcus aureus. The durability of antibacterial wool was assessed and the lysozyme immobilized on wool fabric retained ca. 43% of its activity after five cycles of use when taking the activity of the immobilized lysozyme before using as reference.     

NIK is involved in nucleosomal regulation by enhancing histone H3 phosphorylation by IKKalpha

The exact physiological role of NF-kappaB-inducing kinase (NIK) in the NF-kappaB activation pathway has not been defined, although it is an upstream kinase of IKKalpha. Recent studies have indicated that IKKalpha is a nucleosomal modifier of NF-kappaB signaling. We hypothesized that NIK generates a proximal signal that contributes to IKKalpha modification of nucleosomal structure through phosphorylation of histone H3 and enhancement of target gene expression. By using a chromatin immunoprecipitation assay, our data show that endogenous IKKalpha is recruited to the promoter site of several NF-kappaB-dependent genes in macrophages. Our data show that immunoreactive NIK is rapidly recruited to nuclear compartment in macrophages in response to treatment with endotoxin where it augments phosphorylation of histone H3 by inducing phosphorylation and kinase activity of IKKalpha. A small interfering RNA knockdown of NIK markedly reduces phosphorylation of histone H3 in endotoxin treated macrophages. These data, together, demonstrate a novel role for NIK as a histone H3 modifier, through an accessory pathway from NIK to IKKalpha, that could play an important role in the endotoxin response through modification of nucleosomal structure.     

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimer’s disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48 h after 5% O₂ hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.     

Modification of Bombyx mori silk fabrics by tyrosinase‐catalyzed grafting of chitosan

Tyrosinase could oxidize tyrosyl residues in silk fibroin and result in the production of activated o‐quinone residues, which could facilitate the grafting of the functional amino‐compounds onto silk fibers. In this study, the enzymatic modifications of Bombyx mori silk fibroin with tyrosinase and chitosan were investigated, aiming at improving the properties of silk fabrics, including dyeability, crinkling resistance, and antibacterial activity. The grafting grades of chitosan were evaluated by a color‐development method using bromocresol green. The result indicated that chitosan molecules were not only adsorbed on silk fibers via electrostatic interactions, they also could react with the oxidized silk fibers with tyrosinase. For the silk fabric combinedly treated with tyrosinase and chitosan, tensile strength and crinkling resistance were noticeably increased as compared to that of the chitosan‐treated. The antibacterial activity and its durability measurements revealed the actions of the tyrosinase‐catalyzed grafting of chitosan. The efficacy of the graft reaction might be further enhanced by increasing the accessibility of reactive sites in silk fibers.     

Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase,an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis.